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High temperatures represent a limitation for growth and development of many crop species. Several studies have demonstrated that the yield reduction of tomato under high temperatures and drought is mainly due to a photosynthetic decline. In this paper, a set of 15 tomato genotypes were screened for tolerance to elevated temperatures by cultivating plants under plastic walk-in tunnels. To assess the potential tolerance of tomato genotypes to high temperatures, measurements of chlorophyll fluorescence, pigments content and leaf functional traits have been carried out together with the evaluation of the final yields. Based on the greenhouse trials, a group of eight putative heat-sensitive and heat-tolerant tomato genotypes was selected for laboratory experiments aimed at investigating the effects of short-term high temperatures treatments in controlled conditions. The chlorophyll fluorescence induction kinetics were recorded on detached leaves treated for 60 min at 35 °C or at 45 °C. The last treatment significantly affected the photosystem II (PSII) photochemical efficiency (namely maximum PSII quantum efficiency, Fv/Fm, and quantum yield of PSII electron transport, ΦPSII) and the non-photochemical quenching (NPQ) in the majority of genotypes. The short-term heat shock treatments also led to significant differences in the shape of the slow Kautsky kinetics and its significant time points (chlorophyll fluorescence levels minimum O, peak P, semi-steady state S, maximum M, terminal steady state T) compared to the control, demonstrating heat shock-induced changes in PSII functionality. Genotypes potentially tolerant to high temperatures have been identified. Our findings support the idea that chlorophyll fluorescence parameters (i.e., ΦPSII or NPQ) and some leaf functional traits may be used as a tool to detect high temperatures-tolerant tomato cultivars.
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Plants 2020, 9, 508; doi:10.3390/plants9040508
Eco-Physiological Screening of Different Tomato
Genotypes in Response to High Temperatures: A
Combined Field-to-Laboratory Approach
Carmen Arena
, Stefano Conti
, Silvana Francesca
, Giuseppe Melchionna
, Josef Hájek
Miloš Barták
, Amalia Barone
and Maria Manuela Rigano
Department of Biology, University of Naples “Federico II”, Complesso Universitario Monte S. Angelo, Via
Cintia, 80126 Napoli, Italy;
Department of Agricultural Sciences, University of Naples “Federico II”, Via Università 100, 80055 Portici
(NA), Italy; (S.C.); (S.F.); (G.M.); (A.B.); (M.M.R.)
Department of Experimental Biology, Faculty of Science, Masaryk University, University Campus
Bohunice, Kamenice 753/5, 62500 Brno, Czech Republic; (J.H.); (M.B.)
* Correspondence:; Tel.: +0039-0812532125
Equally contributed.
Received: 31 March 2020; Accepted: 13 April 2020; Published: 15 April 2020
Abstract: High temperatures represent a limitation for growth and development of many crop
species. Several studies have demonstrated that the yield reduction of tomato under high
temperatures and drought is mainly due to a photosynthetic decline. In this paper, a set of 15 tomato
genotypes were screened for tolerance to elevated temperatures by cultivating plants under plastic
walk-in tunnels. To assess the potential tolerance of tomato genotypes to high temperatures,
measurements of chlorophyll fluorescence, pigments content and leaf functional traits have been
carried out together with the evaluation of the final yields. Based on the greenhouse trials, a group
of eight putative heat-sensitive and heat-tolerant tomato genotypes was selected for laboratory
experiments aimed at investigating the effects of short-term high temperatures treatments in
controlled conditions. The chlorophyll fluorescence induction kinetics were recorded on detached
leaves treated for 60 min at 35 °C or at 45 °C. The last treatment significantly affected the
photosystem II (PSII) photochemical efficiency (namely maximum PSII quantum efficiency, F
and quantum yield of PSII electron transport, Φ
) and the non-photochemical quenching (NPQ) in
the majority of genotypes. The short-term heat shock treatments also led to significant differences
in the shape of the slow Kautsky kinetics and its significant time points (chlorophyll fluorescence
levels minimum O, peak P, semi-steady state S, maximum M, terminal steady state T) compared to
the control, demonstrating heat shock-induced changes in PSII functionality. Genotypes potentially
tolerant to high temperatures have been identified. Our findings support the idea that chlorophyll
fluorescence parameters (i.e., Φ
or NPQ) and some leaf functional traits may be used as a tool to
detect high temperatures-tolerant tomato cultivars.
Keywords: heat stress; tomato genotypes; photosynthesis; crop yield; chlorophyll a fluorescence;
Solanum lycopersicum
1. Introduction
Increasing atmospheric temperatures, which are expected to rise by 2–4.8 °C in the next few
decades, can compromise crop productivity in numerous regions worldwide [1–4]. Indeed, elevated
temperatures can induce a series of physiological responses with consequent decreases of crops
Plants 2020, 9, 508 2 of 17
yields and quality [5,6]. Tomato (Solanum lycopersicum), being an excellent source of health-promoting
compounds, is one of the most important crops cultivated worldwide and its heat sensitivity varies
among different genotypes [4,7,8]. Generally, the optimal temperature range for photosynthesis is
considered to be between 25 °C and 30 °C [6]. The rising of average temperatures due to the ongoing
climate change will cause extensive productivity losses in Mediterranean areas, where tomato is
traditionally cultivated [9–11]. In this framework, it becomes important to perform studies that are
able to identify the most promising genotypes able to face heat stress.
The relationship between gas exchange and crop yield has been largely studied in tomato,
suggesting leaf transpiration as the most reliable indicator for yield prediction under drought [12].
However, beside gas exchange, other photosynthesis related parameters [13] should be taken into
account to build a “eco-physiological identity card” for different genotypes.
Chlorophyll fluorescence represents a good tool to rapidly and accurately detect plant health
status, the occurrence of damage within photosystem II (PSII), and to study heat tolerance in vivo
[6,13,14]. The decline of maximum quantum efficiency of PSII (F
), as well as the increase of non-
photochemical quenching (NPQ), are two heat-affected fluorescence parameters [15] related to
photoinhibition and photoprotection mechanisms in response to high temperatures [5,10,16]. Also,
transient changes in fluorescence intensity (Kautsky phenomenon) have been demonstrated to be
particularly suitable for the screening of physiological parameters in plants. The shape of
fluorescence curves changes significantly when plants switch from a healthy status to stress, giving
precious information on plant capability to overcome the stress [17].
Indeed, physiological screening techniques may complement phenotypic measurements and,
therefore, increase the efficiency of the selection of tolerant genotypes [18]. Currently, the majority of
the experiments on tomato responses to heat stress have been carried out in controlled chambers and
only few studies have been performed in the field [11,14,19].
On one hand, with the field approach, it is possible to screen a high number of different
genotypes. On the other hand, it is not easy to separate the effects of heat stress from other
environmental variables, such as light or water depletion, in inducing the plant specific responses
being examined [18]. For these reasons, field experiments should be complemented by laboratory
studies in which the use of novel screening methods, such as chlorophyll fluorescence imaging, may
provide further information for the characterization of tomato genotypes best suited to different
environmental conditions.
This study aimed to evaluate the responses to elevated temperatures in tomato genotypes in a
combined field-to-laboratory approach. First, we investigated the photosynthetic efficiency by
fluorescence emission measurements and leaf structural traits of different tomato varieties grown in
Mediterranean agro-ecosystems during summer in the field. These trials allowed us to identify
functional parameters correlating with final crop yields in different genotypes. After, a group of heat-
tolerant and heat-sensitive tomato genotypes were selected based on crop yield and photosynthesis-
related parameters. These selected genotypes were further characterized in the laboratory, analyzing
the Kautsky fluorescence induction curve after short-term heat treatments to obtain “a signature of
photosynthesis”. More specifically, the shapes of the curves changed when plants were subjected to
stress. The presence and the timing of the appearance of specific fluorescence transients (for
definitions, see [20]) were calculated in order to assess the heat stress-induced changes in PSII
functionality among cultivars.
These laboratory analyses allowed us to validate this easy and very quick protocol as an
alternative method for the selection of potentially heat-tolerant tomato genotypes. The combined
field-to-laboratory approach provides additional information which could help plant biologists and
breeders with characterizing responses to high temperatures in Solanum lycopersicum.
2. Results
2.1. Greenhouse Trials: Correlations Between Physiological Parameters and Yields
Plants 2020, 9, 508 3 of 17
Fifteen tomato genotypes, differing in geographical origin, were grown at supra-optimal
temperatures in order to identify the high and low performers in field environmental conditions. The
genotypes were selected on the basis of crop yield and eco-physiological indices. During anthesis,
maximum air temperatures inside the greenhouse were in the range of 24–43 °C. Significant
differences between each genotype vs. the control genotype JAG8810 were recorded for leaf
functional traits, chlorophylls and carotenoids contents and final yields, as reported in Table 1. The
hybrid JAG8810 had a good yield under high temperatures (from Monsanto, unpublished results).
Therefore, this line may be considered as a positive control which is able to resist extreme high
Table 1. Leaf traits (DW = dry weight of individual leaves, LA = leaf area, SLA = specific leaf area),
total carotenoids, chlorophylls (Chl a and Chl b) and crop yield per plant (YP) evaluated on tomato
genotypes grown under a plastic walk-in tunnel. Values are means ± standard deviation. ANOVA
with Least Significant Difference (LSD) post-hoc test was used to compare each genotype vs. the
control genotype JAG8810. The asterisks indicate statistically significant differences at * p < 0.05; ** p
< 0.01; *** p < 0.001.
(mg 100 g
Chl a (mg 100
Chl b (mg
100 g
(kg pt
E7 0.04 ± 0.10 11.41 ± 1.71 278.17 ± 63.62
* 28.76 ± 0.07 *** 104.82 ± 0.13
36.53 ± 0.29
0.98 ± 0.00
E8 0.10 ± 0.04 14.37 ± 2.86 155.73 ± 36.29 41.30 ± 0.25 *** 169.30 ± 0.27
76.05 ± 0.46
0.77 ± 0.17
E17 0.08 ± 0.02 17.76 ± 2.37 231.60 ± 45.53 44.67 ± 0.23 *** 177.92 ± 0.29
75.18 ± 0.77
1.26 ± 0.17
E36 0.14 ± 0.03
*** 20.09 ± 3.53 ** 146.83 ± 15.50 39.94 ± 0.10 *** 159.32 ± 0.37
67.97 ± 0.17
0.94 ± 0.22
0.15 ± 0.04
*** 13.12 ± 2.52
92.48 ±
17.82** 34.40 ± 0.03 ***
133.61 ± 0.64
54.61 ± 0.64
0.88 ± 0.17
E42 0.10 ± 0.03 18.02 ± 3.11 188.57 ± 37.70 43.02 ± 0.54 *** 174.35 ± 1.62
76.43 ± 0.83
*** 2.25 ± 1.04
E45 0.16 ± 0.02
27.58 ± 4.78
*** 171.16 ± 26.94 36.61 ± 0.04 * 142.81 ± 0.36
*** 59.02 ± 0.35 ** 2.24 ± 1.04
E53 0.07 ± 0.02 12.50 ± 2.19 210.79 ± 62.56 34.37 ± 0.06 *** 132.38 ± 0.10
53.01 ± 0.26
1.32 ± 0.28
E76 0.11 ± 0.01**
21.17 ± 2.25 ** 210.79 ± 62.56 44.63 ± 0.12 ***
171.90 ± 0.06
68.26 ± 0.37
0.64 ± 0.01
E107 0.07 ± 0.01 13.87 ± 2.95 194.36 ± 30.79 36.43 ± 0.04 ** 134.20 ± 0.28
48.49 ± 0.44
0.42 ± 0.12
IL12-4-SL 0.10 ± 0.02 21.00 ± 1.83 ** 217.16 ± 48.16 39.71 ± 0.07 *** 149.58 ± 0.11
56.68 ± 0.20
2.77 ± 0.24
JAG8810 0.07 ± 0.01 13.46 ± 1.86 203.15 ± 55.39 42.96 ± 0.29 161.63 ± 1.51 60.49 ± 1.25 2.99 ± 0.5
LA2662 0.12 ± 0.01
** 21.59 ± 2.07 ** 190.13 ± 24.57 36.98 ± 0.03 *** 143.90 ± 0.42
*** 58.78 ± 0.47 ** 1.97 ± 0.66
LA3120 0.13 ± 0.03 * 20.57 ± 1.14
*** 166.53 ± 36.45 39.30 ± 0.13 *** 155.43 ± 0.23
65.78 ± 0.60
*** 1.91 ± 0.98
M82 0.15 ± 0.03
24.37 ± 4.93
*** 163.26 ± 12.14 32.87 ± 0.14 *** 121.82 ± 0.23
44.88 ± 0.20
*** 3.25 ± 0.6 *
Leaf dry weight (DW) and leaf area (LA) of genotypes E36, E45, E76, LA2662, LA3120 and M82
were significantly higher compared to JAG8810. The genotype E7 was the only one with SLA values
higher than JAG8810. The pigments content (chlorophyll a and b and carotenoids) of all genotypes
was significantly lower than JAG8810, whereas only the M82 genotype showed a crop yield higher
than JAG8810, considered as the control. Chlorophyll fluorescence parameters (F
, Φ
and NPQ)
are shown in Figure 1.
Plants 2020, 9, 508 4 of 17
Figure 1. Maximum quantum yield of photosystem II (PSII) (F
), effective quantum yield of PSII
), and non-photochemical quenching (NPQ) measured on different tomato genotypes grown
under a plastic walk-in tunnel. Bars are means ± standard error (n = 5). ANOVA with LSD post-hoc
test was used to compare each genotype vs. the control genotype JAG8810 (green column). The
asterisks indicate statistically significant differences at * p < 0.05; ** p < 0.01; *** p < 0.001.
Based on field screening, the photochemical parameters differed among genotypes; more
specifically, seven genotypes, namely E8, E17, E32, E37, E42, IL12-4-SL and LA2662, showed higher
values compared to JAG8810. The genotypes E8, E42 and LA2662 also exhibited higher Φ
compared to the control genotype. Conversely, E76 and E107 genotypes showed the highest NPQ
values compared to the control and the other genotypes. The correlations among the analyzed
physiological and structural parameters are reported in Table 2.
Plants 2020, 9, 508 5 of 17
Table 2. Pearson’s correlations between physiological parameters, pigment content and crop yield
production (* p < 0.05; ** p < 0.01). YP = yield per plant; F
= maximum quantum yield of PSII; Φ
= effective quantum yield of PSII; NPQ = non-photochemical quenching; DW = leaf dry weight; SLA
= specific leaf area; LA = leaf area; Chl a = chlorophyll a; Chl b = chlorophyll b; Car = carotenoids.
Chl a
Chl b
−0.647 **
−0.650 **
0.744 **
Chl a
0.970 **
0.983 **
Chl b
0.909 **
Crop yield was negatively correlated with NPQ. Leaf area was positively correlated with leaf
DW. The maximum quantum efficiency of PSII (F
) differed among genotypes, although no
significant correlation was found between crop yield and F
. No correlations were found between
crop yield and chlorophylls or carotenoids content.
Based on crop yield (YP) and NPQ (higher YP and lower NPQ, Table 1 and Figure 1), the top
five performers (genotypes IL12-4-SL, JAG8810, LA3120, LA2662 and M82) along with the low
performer (genotype E107), were chosen for further analyses.
2.2. Chlorophyll a Fluorescence Measurements on Detached Leaves: Heat Shock Treatment at 35 °C and 45 °C
Chlorophyll fluorescence transients and derived parameters were measured as described in the
Materials and Methods section on detached leaves from the selected genotypes and from an
additional two tomato varieties, BG1620 and E41, which are supposedly heat-sensitive from previous
studies carried out in our laboratory.
No significant differences in the chlorophyll fluorescence parameters after a 60-min heat shock
at 35 °C were found between control and treated leaves (data not shown).
Conversely, heat shock at 45 °C for 60 min resulted in significant damage to PSII compared to
control (Figure 2).
Plants 2020, 9, 508 6 of 17
Figure 2. Maximum quantum yield of PSII (F
), effective quantum yield of PSII (Φ
) and non-
photochemical quenching (NPQ) in detached tomato leaves of different tomato genotypes following
short term heat treatment for 60 min at 45 °C (solid bars), compared with the respective non-treated
control (open bars). Bars are means ± standard error (n = 5). The asterisks indicate statistically
significant differences (* p < 0.05; ** p < 0.01; *** p < 0.001) according to Student’s t-test.
The short-term heat shock treatment led to a reduction in maximum PSII quantum efficiency
) in almost all genotypes. Interestingly, the highest reductions in F
were recorded in the
two supposedly heat-sensitive genotypes, BG1620 (−52%) and E41 (−19%), that were more affected
by heat stress compared to the genotype LA3120 (−12% F
reduction). Contrastingly, the F
ratio was little or not affected by the heat shock treatments in M82 (−4%) and LA2662 (0%) genotypes,
which were among the top performers in the field trial. The quantum efficiency of PSII electron
) was also affected by the heat shock treatment. For most genotypes, a significant
reduction in the Φ
was recorded and LA3120, BG1620, and E41 were found to be the most sensitive
genotypes with a decrease of −46%, −45% and −42% compared to the control, respectively. The M82
and IL12-4-SL genotypes were little or not affected by the heat shock treatment. As a consequence of
Plants 2020, 9, 508 7 of 17
the heat shock treatment, the NPQ values increased significantly in most genotypes compared to their
respective controls, but not in BG1620, IL12-4-SL and M82.
2.3. Heat-Induced Changes in Shape of Kautsky Kinetics
The shape of slow Kautsky kinetics and the derived parameters clearly showed that the effects
of heat treatment vary with genotype. As an example, in Figure 3, the slow Kautsky kinetics of the
least heat sensitive (IL12-4-SL) and the most heat sensitive (E107) genotype are reported. Heat
treatment leads to a reduction of the P peak in both genotypes compared to non-stressed controls.
This decline appears more pronounced for the heat sensitive genotype E107. The comparison among
genotypes showed that the M and S chlorophyll fluorescence signals were missing in heat-treated
samples and resulted in the absence of P/S, P/M, and S/M ratios compared to respective controls
(Table 3).
Figure 3. The effect of heat shock (60 min at 45 °C) on the shape of slow Kautsky kinetics in tomato
plants. Non-treated controls are indicated by open symbols (), heat shock-treated leaves by solid
symbols (). Less sensitive (IL12-4-SL) and substantially sensitive genotypes (E107) are presented.
The curves are means of at least 4 replicates (leaves). Data are normalized to background chlorophyll
fluorescence (F
). The chlorophyll fluorescence levels: O, fluorescence minimum; P, fluorescence peak;
S, semi-steady state; M, fluorescence maximum; T, terminal steady state are indicated.
Plants 2020, 9, 508 8 of 17
Table 3. Heat-induced changes in chlorophyll fluorescence parameters characterizing the shape of slow
Kautsky kinetics recorded for 8 tomato genotypes. Values are means of 5 replicates. Standard deviation
(not shown here) was under 4% of means. Asterisks indicate the statistical significance of the difference in
heat treated leaves compared to their respective non-treated control (* 0 to 50%; ** 50 to 100%; *** over
100%). O (origin) = minimum fluorescence level (also termed F
); P = peak fluorescence level reached after
1–2 s of actinic light exposure; S = semi-steady state of fluorescence emission; M = maximum of fluorescence;
T = terminal steady state chlorophyll fluorescence of the slow Kautsky kinetics; Fp = fluorescence peak; Fs
= fluorescence steady state; Rfd = relative fluorescence decline (vitality index); t
= time at which P
fluorescence peak is reached; t
= time at which S fluorescence level is reached; t
= time at which M
fluorescence level is reached; t
= time at which T fluorescence level is reached, Dip = decrease.
BG1620 E41 E107 IL12-4-SL JAG8810 LA2662 LA3120 M82
O/P 0.229 0.242 0.212 0.220 0.201 0.239 0.194 0.207
P/S 1.810 1.167 1.065 1.517 1.405 1.242 1.193 1.673
S/M 0.922 - 0.994 0.916 0.938 - 0.975 0.888
P/T (steady state) 2.665 3.101 2.101 2.482 2.428 2.887 2.694 2.801
M/T (steady state)
Rfd = (Fp−Fs)/Fs 1.665 2.101 1.101 1.482 1.428 1.887 1.694 1.801
6.080 3.360 4.080 4.080 4.080 4.080 −29.940 6.080
10.080 - 2.720 10.080 8.080 - 6.080 12.080
270.060 270.060 270.060 270.060 270.060 270.060 270.060 270.060
Dip at 26.080 - 52.080 42.080 - 48.080 86.080 38.080
Heat Treated (60 min at 45 °C)
0.713 ***
0.483 **
0.341 **
0.344 **
0.366 **
0.252 *
0.379 **
0.316 **
1.280 *
0.936 *
0.855 *
P/M - - - 1.166 * 0.929 * - - 0.853 *
S/M - - - 0.911 * 0.993 * - - 0.997 *
P/T (steady state) 1.658 * 2.729 * 2.680 * 2.250 * 2.407 * 2.779 * 2.881 * 2.302 *
M/T (steady state) - - - 1.929 * 2.591 * - - 2.700 *
Rfd = (Fp-Fs)/Fs
0.658 **
1.729 *
1.680 **
1.250 *
1.407 *
1.779 *
1.881 *
1.302 *
1.280 * 1.280 * 1.760 ** 1.120 * 1.280 * 1.040 1.280 1.040
- - - 4.080 2.240 *** - - 3.360
- - - 8.080 2.400 *** - - 4.080
255.060 * 270.060 * 245.060 * 260.060 * 245.060 * 260.060 * 270.060 * 250.06 *
Dip at
34.080 *
40.08 *
It may be generalized that for all tomato genotypes the shape of the slow Kautsky kinetics was
affected mainly during the early phase (i.e., within the first 60 sec after the actinic light was switched
on). The most pronounced change was found in the O/P ratio in BG1620 which showed a relative
change of about 200%, while other genotypes showed a much lower variation (LA2662: 5.4%). Apart
from the shape of the slow Kautsky kinetics and ratio values, the time at which the P, S, and M points
were reached differed within treatments. Heat treatment led to an increase in t
in four cultivars.
However, t
showed no change in three genotypes, and a decrease in the IL12-4-SL genotype.
3. Discussion
To date, there is a lack of knowledge about how differently sensitive/tolerant tomato genotypes
would respond to heat events. In heat-sensitive tomato genotypes, high temperatures are responsible
for the decrease in photosynthesis and overall crop yield [6].
In this paper, a set of eco-physiological parameters have been proposed to screen the most
promising tomato genotypes to be cultivated under elevated temperatures. A combined
field/laboratory experimental approach was performed. Firstly, a field trial was carried out under a
plastic walk-in tunnel to assess the field performances of tomato genotypes cultivated at high
temperatures (up to 43 °C) in terms of crop yield and physiological traits. In the second part of this
study, the heat-resistant and heat-sensitive genotypes were tested in the laboratory to analyze their
Plants 2020, 9, 508 9 of 17
responses to short-term heat shock and to investigate the photochemical behavior related to their
different field performance.
We suppose that at the temperatures considered in this study, the photosynthetic apparatus was
not damaged but rather regulated in the different genotypes, contributing to the degree of their
sensitivity or resistance to heat.
Our field studies indicated that tomato genotypes with higher yields also had lower NPQ values.
Non-photochemical quenching is considered a key mechanism in photoprotection against light and
temperature stress in higher plants [16,21]. High NPQ is often associated with conformational
changes within PSII, which transiently depresses CO
fixation [22].
In our trials, the top performers in terms of yield (genotypes IL12-4-SL, M82, LA2662, LA3120
and JAG8810) also showed the lowest NPQ values. Our data suggest that under experimental field
conditions, these tomato genotypes are more efficient in transferring the light excitation energy to
fixation, thus producing a higher photosynthetic carbon gain. Contrastingly, the highest NPQ
values measured in the low performing genotypes (E37, E76 and E107) correspond to more intense
thermal energy dissipation, leading to lower CO
assimilation and crop yield.
In heat-tolerant tomato genotypes, the NPQ protection is likely activated less promptly than in
the heat-sensitive genotypes. Conversely to NPQ, the F
ratio and Φ
did not show any
correlation with crop productivity, suggesting that the higher F
values in some genotypes may
indicate a better photosynthetic performance that is not always related to a higher crop yield. These
data are in contrast with findings of many authors, who demonstrated that the F
ratio is one of
the fluorescence parameters most affected by high temperatures and used it as an index for screening
tomato genotypes under heat stress [14,15,23]. Indeed, our data indicated that the high temperatures
experienced by plants in the field did not compromise the quantum yield of PSII in any genotype.
Based on the results of Hückstädt et al. [24], it may be hypothesized that the detrimental effect
of the high diurnal temperatures on photosystems has been compensated for by the optimal
temperatures [6] reached in the greenhouse during the night (see Figure 4).
Elevated temperatures could also determine a loss in the amount of photosynthetic pigments.
Therefore, the capacity of some genotypes to maintain higher pigments content under heat stress, as
well as to adjust some leaf functional traits (i.e., SLA, LA, RWC), are considered key heat tolerance-
linked traits [6,25–27].
For example, a higher SLA can be essential to obtain a higher potential evaporative demand and
a more extensive foliar display to capture more light [26]. However, in this work, no significant
correlation was found between final crop yield and pigments content or final crop yield and SLA,
indicating that these traits are not automatically linked to crop productivity. Therefore, these traits
were not considered for the selection of the heat-tolerant and heat-sensitive genotypes in this study.
On the basis of field trials, the tomato genotypes IL12-4-SL, JAG 8810, LA3120, LA2662 and M82
were selected as the top performers in terms of yield and low NPQ values, whereas the genotype
E107 was selected as a low performer considering the low photochemical efficiency and crop yield.
The heat sensitive BG1620 and E41 genotypes were added to the list of genotypes to be further studied
in the laboratory. For these tomato genotypes, the chlorophyll fluorescence transient analysis (slow
Kautsky kinetics) was performed in response to heat treatments to separate the effects of the high
temperature from other environmental constraints in the field.
In a previous study on tomato, plants exposed to 42 °C for 24 h showed a decline of net
photosynthesis, maximum PSII photochemical efficiency and electron transport rate [28]. Consistent
with these findings, our results further demonstrated that a short-term (60 min) heat shock at 45 °C
was sufficient to cause significant effects on the photochemistry in detached tomato leaves.
Interestingly, the F
ratio was found to be most affected in the heat-sensitive genotype BG1620
and in the low performer E107 genotype. An F
reduction of only 8% was registered in the
genotype LA2662, which was selected as heat-tolerant in the field experiment. These data are also in
agreement with Sharma et al. [18] who measured a reduction of F
in detached wheat leaves at 45
°C and Camejo et al. [29] who reported an F
decrease in a heat-susceptible tomato cultivar and
no changes in heat-tolerant cultivar.
Plants 2020, 9, 508 10 of 17
We supposed that the F
and PSII decreases in the sensitive (BG1620 and E41) genotypes
could be due to heat-induced structural modifications in PSII, particularly D1 protein oxidative
degradation. It has been demonstrated that heat stress may cause cleavage of the reaction center-
binding protein D1 and induce dissociation of a manganese-stabilizing 33 kDa protein from the PSII
reaction center complex [30]. Such oxidative damages have a strong positive relationship with the
accumulated levels of reactive oxygen species (ROS) and lipid peroxidation under heat stress [31].
However, it cannot be excluded that the significant reduction of F
observed in some
genotypes may also be associated with photoprotection mechanisms, as indicated by NPQ values
that peaked in correspondence to the reduction in F
. Simultaneously, protective mechanisms are
activated to protect the D1 protein, such as the expression of heat shock proteins (HSPs) like HSP21
that directly binds D1 to shield it against damage [32]. However, this does not seem to be the case for
the genotype BG1620, which was the most affected by the severe decline of photochemical and non-
photochemical processes.
The decrease in F
values in detached tomato leaves that were heat-treated at 40 °C was also
reported by Willits and Peet [33]. In our work, a heat shock treatment at 35 °C on detached leaves
was similarly tested with no significant effects on the photochemical efficiency of PSII (data not
shown), supporting the idea that such responses depend on the severity of the heat stress applied
The analysis of the shape of the slow Kautsky kinetics and the parameters calculated from its
significant time points (chlorophyll fluorescence levels O, P, S, M, T) revealed that heat shock
treatment led to significant differences compared with the control. Polyphasic changes in chlorophyll
fluorescence signal in the PSMT part of the Kautsky kinetics represent the combined effect of
photochemical and non-photochemical processes taking place in the chloroplast [34]. As the main
changes happened during the first 60 s of actinic light exposure, they might be attributed to the
interperiod of balancing the rate of primary photochemical processes in PSII to the rate of CO
Since the S and M chlorophyll fluorescence levels were generally missing in heat-treated tomato
leaves, the processes responsible for S and M interstates (i.e., the processes regulating the Calvin–
Benson cycle of CO
fixation, such as limitations in NADP+, phosphate pool equilibration, and
transmembrane ΔpH formation [20]) were overwhelmed by a strong non-photochemical quenching
that was activated by the heat shock treatment. This may be a consequence of the heat-induced
thermal dissipation of absorbed light energy, such as state 1 to state 2 transitions causing preferential
excitation of PSI and structural changes in thylakoid membranes, as reported by Marutani et al. [35].
The activation of these protective mechanisms may, however, lead to PSI damage in high temperature
treated plants due to over-reduction of the acceptor side of PSI [13].
The changes observed during the transition from P to S phase of the Kautsky kinetics indicate
the actual proportion between the mechanisms involved in photochemical and non-photochemical
quenching [36]. Since the parameters derived from the slow Kautsky kinetics responded to heat
treatment, it might be concluded that they have a high potential in the evaluation of heat effects on
the chloroplast function of tomato, as shown for light stress and leaf age effects by Nesterenko et al.
[37]. Many studies support the idea that a sustained increase in leaf photosynthesis can also lead to
an increase in total biomass production [38].
Overall, in this work, several useful photosynthetic parameters were identified, which could be
essential to detect and describe high temperature-tolerant tomato cultivars. These parameters could
be used as an effective tool for the prompt identification of tomato genotypes tolerant to high
4. Materials and Methods
4.1. Plant Material and Growth Conditions
Fifteen genotypes have been tested in the field. Of these genotypes, eleven genotypes (marked
as E## in Table 4) were selected based on their different productivity (demonstrated in a previous
Plants 2020, 9, 508 11 of 17
experiment conducted in the Campania region in the year 2016 [39]). The genotypes JAG8810
(Monsanto F
hybrid), LA2662 (Saladette) and LA3120 (Malintka) were reported to have high fruit
productivity under high temperatures (JAG8810, from Monsanto, unpublished results; LA2662 and
LA3120, Tomato Genetic Resources Center [40]). The JAG8810 hybrid may be considered as a positive
control which is able to resist extreme high temperatures. The M82 and the IL12-4-SL genotypes,
previously selected and characterized in a recent paper [41], were added to the list of genotypes to be
tested because their physiological response to elevated temperatures was unknown. One additional
tomato variety, BG1620 (kindly provided by Prof. G. Pevicharova, MVRCI Bulgary), was also added
to the set of genotypes to be analyzed. The genotypes BG1620 and E41 are supposedly heat-sensitive
based on previous analyses carried out in our laboratories (unpublished data).
Table 4. Tomato genotypes analyzed in this study.
No. Genotype Origin Common Name
1 E7 Italy Corbarino PC04
Corbarino PC05
3 E17 Italy Pantano Romanesco
4 E36 Italy Riccia San Vito
5 E37 Italy Siccagno
7 E42 Italy PI15250
8 E45 Italy SM246
9 E53 South America Latin American cultivar (Honduras)
Black Plum
11 E107 Europe E-L-19, Spain
12 JAG8810 - Monsanto F1 hybrid
13 M82 California M82
15 LA2662 - Saladette
16 LA3120 - Malintka
17 BG1620 Bulgary -
Tomato plants were grown in the year 2017 in Battipaglia (Salerno, Italy) (40°23′03 N, 17°21′17
E, 72 m a.s.l.) in a Mediterranean or Csa climate according to the Köppen classification scheme [42],
under walk-in thermal polyethylene tunnels. During the whole cultural cycle, climatic data were
recorded using the weather station VantagePro2 from Davis Instrument Corp. The maximum and
minimum temperatures during anthesis are reported in Figure 4. Spatial variation in temperature
within the walk-in tunnels was found to be minimal.
Plants 2020, 9, 508 12 of 17
Figure 4. Relative humidity (R.H.; open symbols, right axis) and maximum and minimum air
temperatures (solid symbols, left axis) during May–July 2017 inside a greenhouse in the experimental
field at Battipaglia (Salerno, Campania region, Italy).
The seeds of all genotypes were first rinsed and soaked in distilled water and then kept for 4
days in 8.5 cm diameter Petri dishes over 3 layers of filter paper saturated with distilled water. After
germination, the seeds were sown in seed trays kept in the greenhouse. Seedlings were transplanted
in April under plastic walk-in tunnels. Plants were grown following the standard cultural practices
of the area. Insecticides and fungicides were applied to the plants according to general local practices
and recommendations. Urea phosphate fertilizer (40 kg ha
) was applied to the soil before
transplanting. Tillage treatments included plowing that was followed by one/two milling. Weeding
and ridging were also carried out. Through fertirrigation, recommended levels of N (190 kg ha
) and
K (20 kg ha
) were applied. During cultivation, plants were irrigated as required.
All genotypes were grown according to a completely randomized experimental design with
three replicates and 10 plants per replicate. Total fruit number and fresh weight were measured at the
end of growth season to evaluate the crop yield per plant (YP). The crop yield was measured at the
red fruit ripe stage.
4.2. Functional Leaf Trait Analysis
The measurements of leaf area (LA), specific leaf area (SLA) and leaf dry weight (DW) were
performed on the fourth leaf from the apex in each plant. Five leaves for each genotype were sampled
from 5 different plants. LA was measured using ImageJ 1.45 software for image analysis [43]. The
leaves were then dried at 70 °C and their DW was measured after 48 h. SLA was calculated as the
ratio of leaf area to leaf dry weight and expressed as cm
DW according to Cornelissen et al. [44].
4.3. Chlorophyll Fluorescence Emission Measurements in the Field
Chlorophyll fluorescence parameters were measured on fully expanded leaves (the fourth leaf
from the apex) using a portable FluorPen FP100 Max fluorometer, equipped with a
Photosynthetically Active Radiation (PAR) sensor (Photon Systems Instruments, Drásov, Czech
Republic) following the procedure reported by Sorrentino et al. [45]. Five replicate measurements for
each genotype were taken as follows.
The ground state fluorescence (F
) was induced by an internal Light Emitting Diode (LED) blue
(1–2 μmol photons m
) on 30-min dark-adapted leaves of plants moved into a dark room. The
maximum fluorescence level in the dark-adapted state (F
), was triggered by a 1 s saturating light
pulse of 3000 μmol photons m
, and the maximum quantum efficiency of PSII (F
) was
calculated according to Equation (1).
The fluorescence readings in the light were taken using an open leaf-clip, allowing for
measurements of the steady state fluorescence level (Fs) at an ambient light Photosynthetic Photon
Flux Density (PPFD) of 150–200 μmol m
in the PAR spectrum and of the maximum fluorescence
level in light-adapted leaves (F’
) measured after a saturating light pulse.
The quantum yield of PSII electron transport
) was calculated according to Equation (2)
[46]. Non-photochemical quenching (NPQ) was calculated using Equation (3) [47].
= (F
− F
= (F’
− F
NPQ = (F
− F’
4.4. Determination of Total Chlorophylls and Carotenoids Content
Following the chlorophyll fluorescence field measurements, the same leaves were excised,
stored in a cool box and transferred to the laboratory for the determination of photosynthetic
pigments content (chlorophyll a, chlorophyll b and carotenoids) according to the method described
Plants 2020, 9, 508 13 of 17
by Rigano et al. [5]. One gram of leaf sample was extracted with 16 mL of acetone/hexane (40/60, v/v)
with a T-25 Ultra-Turrax Homogenizer (IKA-Werke GmbH & Co. KG, Staufen, Germany). The
homogenate was then centrifuged at 5000 rpm for 5 min at 4 °C and supernatants were collected and
stored at −20 °C prior to spectrophotometric analysis. Pigment contents were calculated in mg on 100
g of leaf fresh weight. Three separate biological replicates for each sample and three technical assays
for each biological repetition were measured.
4.5. Chlorophyll a Fluorescence—Heat Treatments and Laboratory Measurements
Chlorophyll fluorescence transients and numeric parameters were measured with a FluorCam
(Photon Systems Instruments, Brno, Czech Republic) on detached leaves from genotypes selected
from the in-field analyses. Two additional tomato varieties, BG1620 and E41 (supposedly heat-
sensitive based on previous analyses carried out in our laboratories (unpublished data), were also
added to the set of genotypes to be studied in this research.
Whole compound leaves from field grown tomato plants were sampled in the morning, between
2 and 3 h after dawn (8:00 to 9:00 a.m.). Fully expanded leaves (the fourth leaf from the apex) were
excised at the base of the petiole from each plant using a sharp blade and the cut base was
immediately immersed in distilled water in a 50 mL test tube in order to prevent dehydration. The
sampled leaves were then temporarily stored in a dark cool box and transferred to the laboratory for
the short-term high temperature treatments and chlorophyll fluorescence experiments.
In the laboratory, single leaflets were excised from the tomato compound leaves and placed in
9.0 cm diameter Petri dishes over water saturated filter paper. Two groups of leaf samples were
selected for each genotype: one group was kept at the laboratory room temperature (25–26°C) in the
dark and assumed as control treatment, while another group was placed in a thermostatic cabinet
set, either at 35°C or at 45°C, for 1 h in the dark and was considered the heat treatment. At the end of
the heat treatment, leaf samples were adapted at room temperature for 20 min in the dark prior to
performing the fluorescence measurements. The same procedure was replicated on different leaves
and repeated for each tomato genotype. The whole experiment was carried out subsequently on leaf
samples treated either at 35 °C (lower heat stress level) or at 45 °C (higher heat stress level), plus the
respective controls.
The temperature conditions in this experiment were chosen to represent the optimal
temperature range for tomato (within 25 – 30 °C [6]) and the maximum air temperature of 33–43 °C
encountered by tomato genotypes in the walk-in tunnels during the experimental period.
Chlorophyll fluorescence transients (slow Kautsky kinetics supplemented with quenching analysis)
were measured with a Handy Fluor Cam FC-1000H imaging fluorimeter (Photon Systems
Instruments, Drásov, Czech Republic) controlled by the FluorCam7 software (Photon Systems
Instruments, Drásov, Czech Republic).
The experimental protocol started with the measurement of the ground state (minimum)
fluorescence level (F
, O) when the samples were exposed to low intensity measuring light flashes,
followed by a saturating pulse of light (960 ms, 2400 μmol m
) to induce maximum chlorophyll
fluorescence (F
, P). After 27 s of dark adaptation, the samples were exposed to actinic light (200
μmol m
) for 5 min until steady state chlorophyll fluorescence (F
) was reached. At this point,
another saturating pulse of light induced the maximum chlorophyll fluorescence of the light-adapted
sample (F’
). The maximum quantum efficiency of PSII (F
), the quantum efficiency of PSII
electron transport
) and non-photochemical quenching (NPQ) were calculated using the
FluorCam7 software, according to Equations (1) – (3) reported above, respectively.
4.6. Analysis of Kautsky Kinetics Shape in Response to Heat Treatment
Slow Kautsky kinetics is routinely used to evaluate the sensitivity of plants to a wide variety of
stressors [48,49]. In this study, the analysis of kinetic fluorescence shape, i.e., the presence and the
time of appearance of specific fluorescent transients (O, P, S, M and T), was utilized to assess heat
stress-induced changes in PSII functionality in tomato. For individual Kautsky kinetics recorded after
a heat treatment (see above), chlorophyll fluorescence levels O, P, S, M and T were identified, as well
Plants 2020, 9, 508 14 of 17
as the times at which they were reached. Effects of experimental temperature on the O-, P-, S-, M-,
and T-derived parameters were then evaluated for the individual genotypes and the genotype-
dependent responses of the parameters were characterized.
4.7. Statistical Analysis
Statistical analysis was performed on all measured traits using SPSS 23 Software (IBM SPSS
Statistics, USA). Analysis of variance (ANOVA) was used to check for significant differences between
each genotype vs. the control genotype (JAG8810) and where significant differences were found, the
least significant difference (LSD) at the 0.05, 0.01 or 0.001 level of probability was calculated and used
to compare the mean values. Student’s t-test was performed to check for differences between control
and heat-treated samples in the case of detached leaf experiments. Pearson’s correlation coefficient
was used to test associations between tomato yield and other variables.
5. Conclusions
Due to ongoing climate change, the screening and identification of the most promising tomato
cultivars able to maintain elevated productivity under heat stress becomes a priority for farmers and
producers to avoid significant losses of crop yield.
In our experiments, heat tolerant and heat sensitive tomato cultivars were identified and
characterized using a correlative approach combining different field and laboratory methods based
on functional leaf traits, crop yield and photochemical indexes.
The three main outcomes emerging from this work include the confirmation that some
parameters linked to chlorophyll fluorescence emission can be used to phenotype heat tolerance in
tomato, both in the field and in the laboratory. Secondly, we demonstrated that the detached leaf
method can be used as an easy, quick and valid alternative for the selection and characterization of
potentially heat-tolerant tomato genotypes. The advantage of a laboratory approach that implements
field measurements is that it is possible to separate the effects caused by heat treatments from the
other related environmental factors such as high light, low relative humidity and limited water
supply. Finally, we identified five tomato genotypes (JAG8810, LA3120, LA2662, IL12-4-SL and M82)
as promising genotypes that are potentially tolerant to elevated temperatures. These genotypes also
represent a valuable resource to be used in future works aiming to assess the underlying
physiological mechanisms for variability in photosynthetic responses among different cultivars.
Author Contributions: Conceptualization, C.A., S.C., A.B. and M.M.R.; Data curation, C.A., S.C., S.F., J.H., M.B.
and M.M.R.; Funding acquisition, A.B.; Investigation, C.A., S.C., S.F., G.M., J.H., M.B. and M.M.R.; Writing—
original draft, C.A., S.C., M.B., A.B. and M.M.R.; Writing—review and editing, C.A. and M.M.R. All authors
have read and agreed to the published version of the manuscript.
Funding: The authors have received funding from the European Union’s Horizon 2020 research and innovation
programme through the TomGEM project under grant agreement no. 679796.
Conflicts of Interest: The authors declare no conflict of interest.
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... However, the specific feature of arid climate is low water content in both soil and air. The temperature increase goes hand in hand with the decrease of air relative humidity (RH) (Arena et al., 2020). Lower RH of air should stimulate gas exchange between leaves and environment. ...
... We found the only work specifying that control plants were kept at 70 ± 7% RH and experimental plants were heated at 48 ± 6% RH (Sharma et al., 2015). Before the HS experiments, plants were grown in the conditions varied from 40% RH (Pastenes and Horton, 1996;Dogru, 2021) to ~90% RH (Arena et al., 2020); more often plants were grown at the conditions 60-65% RH (Havaux, 1996;Yan et al., 2013;Turan et al., 2019;Ferguson et al., 2020;Yudina et al., 2020). So, we can not be sure whether these fluctuatuions are critical or not. ...
... The longer application of HS brings the protective mechanisms of the cellular level into the analyses, for example, the de novo synthesis of photosynthetic polypeptides that requires all the steps of gene expression from transcription (Zubo et al., 2008) to translation (Franco et al., 1999;Tanaka et al., 2000). The prolonging HS to several hours -1h (Arena et al., 2020), 2h (Camejo et al., 2005;Yan et al., 2013), and 3h (Kalituho et al., 2003) is less popular. Applying HS for days or many hours at least allows studying protective mechanisms at the level of a whole plant. ...
The warming is global problem. In natural environments, heat stress is usually accompanied by drought. Under drought conditions, water content decreases in both soil and air; yet,the effect of lower air humidity remains obscure. We supplied maize and barley plants with an unlimited source of water for the root uptake and studied the effect of relative air humidity under heat stress. Young plants were subjected for 48 h to several degrees of heat stress: moderate (37 °C), genuine (42 °C), and nearly lethal (46 °C). The conditions of lower air humidity decreased the photochemical activities of photosystem I and photosystem II. The small effect was revealed in the control (24 °C). Elevating temperature to 37 °C and 42 °C increased the relative activities of both photosystems; the photosystem II was activated more. Probably, this is why the effect of air humidity disappeared at 37 °C; the small inhibiting effect was observed at 42 °C. At 46 °C, lower air humidity substantially magnified the inhibitory effect of heat. As a result, the maximal and relative activities of both photosystems decreased in maize and barley; the photosystem II was inhibited more. Under the conditions of 46 °C at lower air humidity, the plant growth was greatly reduced. Maize plants increased water uptake by roots and survived; barley plants were unable to increase water uptake and died. Therefore, air humidity is an important component of environmental heat stress influencing activities of photosystem I and photosystem II and thereby plant growth and viability under severe stress conditions.
... Chlorophyll fluorescence analysis was performed in vivo on 5 leaves per 3 plants per both HD and LD plots, by using a portable pulse amplitude-modulated fluorometer (Fluor-Pen FP 100max), equipped with a light sensor (Photon System Instruments, Brno, Czech Republic) following the procedure reported in Arena (2020) [41]. The basal fluorescence (F 0 ) was induced on 30 min dark-adapted leaves by an internal blue light pulse of about 1-2 µmol photons m −2 s −1 . ...
... Furthermore, the values of Fv/Fm between 0.79-0.84 also confirmed a healthy status for plants with no signal of stress [41,59]. However, a more fine analysis revealed that some differences occur between HD and LD leaves as regards other photochemical indexes, namely ΦPSII, ETR, and NPQ. ...
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In the Mediterranean region, some areas of the Vesuvius National Park (southern Italy) are subjected to a severe anthropogenic impact, especially during spring and summer seasons. The continuous trampling of tourists and buses leads to the formation of “dust-clouds”, exposing plants, especially along the paths, to a great deposition of powder particles on leaves. The aim of this study was to analyze if the dust deposition induces changes in leaf morpho-anatomical and eco-physiological traits of the alien, invasive, species Robinia pseudoacacia L., with particular attention to the photosystem II (PSII) efficiency. We selected plants located near the paths with a high deposition of dust (HD) and plants far away from the paths (low deposition, LD), and tested them over three dates along summer. We analyzed PSII photochemistry, photosynthetic pigments content, and leaf functional (e.g., relative water content and leaf dry matter content) and morpho-anatomical traits (e.g., parenchyma thickness, mesophyll density). HD plants presented a more efficient PSII activity, indicated by the higher quantum yield of PSII electron transport (FPSII) (9%) and electron transport rate (ETR) (38%) in the end of July. Dust deposition also reversibly altered photosynthetic pigments concentration and some lamina traits, adjustable in the short-term (e.g., intercellular spaces and phenolics distribution). We hypothesize that HD leaves were shielded by dusts which would protect their photosynthetic apparatus from the excess of light.
... Developing new and improved tomato genotypes is more vital than ever to maintaining tomato production and ensuring global food security, especially as the global population grows rapidly and the climate changes abruptly. The lack of genetic diversity and the unavailability of high-yielding cultivars are the main reasons for low yield [9][10][11][12]. Accordingly, assessing genetic variability is essential for developing new genotypes with the desired combination of traits [13][14][15]. Exploring genetic diversity becomes decisive before planning an appropriate breeding strategy for genetic improvement [16,17]. ...
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Tomato is the most consumed vegetable crop worldwide, with excellent beneficial health properties and high content of vitamins, minerals, carotenoids, total antioxidants, and phenolic compounds. Hence, improving its genotypes is crucial to sustain its production and ensure food security, principally under the fast-growing worldwide population and abrupt global climate change. The present study aimed to explore the genotypic variability associated with specific characteristics in twenty-five diverse tomato genotypes. In addition, the relationships between growth, yield, and quality traits using both univariate (correlation coefficient, path analysis) and multivariate (principal component, principal coordinates, canonical variate) analysis methods were explored. The results indicated that the evaluated genotypes possessed highly significant variation. This is appropriate for future hybridization through tomato breeding programs. All evaluated genotypes demonstrated considerable potential to develop strong hybrid vigour for growth, yield, and quality characteristics. In particular, the genotypes LS009, LS011, and LS014 could be considered promising, high-yielding, and resistant to yellow leaf curl virus infestation (YLCV) disease parents for future breeding schemes. The number of fruits per plant, fruit diameter, and fruit weight proved strong positive relationships with fruit yield. Accordingly, these characteristics demonstrate their importance in improving fruit yield and could be exploited as indirect criteria for selecting high-yielding tomato genotypes through breeding programs.
... The results of this study also imply that melatonin could regulate the balanced flow of electrons in PSII, which prevents chlorophyll pigment degradation and decreases thylakoid membrane damage (F 0 /F m ), which could upregulate the PSII photochemistry and therefore enhance photosynthesis. Similarly, the results of Arena et al. [76] follow the same trend. ...
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In tomato (Lycopersicon esculentum L.), the effects of combined drought (D) and high temperature (HT) stress during the flowering stage had not been studied in detail. Therefore, this study was conducted with an objective of quantifying the effects of foliar spray of melatonin under individual and combined drought and HT stress. At flowering stage, D stress was imposed through withholding irrigation, while HT stress was imposed through exposing the plants to ambient temperature (AT) along with an increase of +5 °C. Under D + HT, plants were first subjected to drought followed by a + 5 °C increase in AT. The duration of individual or combined stress was ten days. At 80% available soil moisture, 100 µM melatonin was sprayed on D, HT, or D + HT treated plants. Among the stresses, D + HT stress increased the thylakoid membrane damage and decreased the photosynthetic rate and fruit yield more than D or HT stress. Foliar spray of 100 µM melatonin produced decreased thylakoid membrane damage [D: 31%, HT: 26%, and D + HT: 18%] and increased antioxidant enzyme, viz., superoxide dismutase, catalase, peroxidase, ascorbate peroxidase, and glutathione reductase, activity over stress-control plants. The photosynthetic rate [D: 24%, HT: 22%, and D + HT: 19%] and fruit yield [D: 32%, HT: 23%, and D + HT: 16%] were increased over stress-control plants. Hence, it is evident that the increased photosynthetic rate and fruit yield in D + HT and 100 µM melatonin-sprayed plants may be associated with an increased antioxidant defense system. Melatonin as a novel biostimulator has a great potential in scavenging free radicals through increased antioxidant activity, which shields the photosynthetic membrane from damage and therefore helps in stress mitigation.
... This problem is exacerbated in greenhouse crops, where temperature increase during summer months is more pronounced. Tomato yield reduction under high temperatures is mainly attributed to a photosynthetic and pollen viability decline [14,15]. This leads to a reduced fruit set and the subsequent decrease in fruit number and size. ...
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Chitosan is a natural polymer with multiple applications in agriculture due to its ability to stimulate plant growth and resistance to both biotic and abiotic stressors. The impact of chitosan application on fruit production and quality was studied under greenhouse conditions in a summer crop in a semi-arid climate. Treatments consisted of the spray application of this biostimulant to the aerial plant part at different doses (0, 0.1, and 1 g L−1). Treatment with the lowest dose did not produce significant differences in yield (total production, number, and mean weight of the fruit), but increased the concentration of flavanones (trusses 2 and 7) and phloretin-C-diglucoside (truss 2) with regard to the control. On the contrary, the high-dose treatment increased the yield due to the rise in the number of fruits and produced a significant decrease in the concentration of vitamin C, lutein, β-carotene, and hydroxycinnamic acids (trusses 2 and 7); lycopene, phytoene, and phytofluene in truss 2; and flavanols and phloretin-C-diglucoside in truss 7. These results show the ability of chitosan to improve tomato yield or to enhance the accumulation of bioactive compounds (phenolic compounds) in fruit, depending on the dose. Results are explained on the basis of the ability of chitosan to activate yield and secondary metabolite production, the dilution effect due to an increased fruit load, and the interaction of chitosan with changing environmental factors throughout the crop cycle.
... Chlorophyll fluorescence (ChlF) parameters were measured on randomly selected hydroponically growing A. thaliana and N. tabacum plants, (at least 5 plants of each treatment), before harvest. The standard protocol of slow Kautsky kinetics supplemented with the saturation pulse method (for details see e.g., Arena et al., 2020) was applied using an FC 800-O/1010 fluorometric camera system (Photon Systems Instruments). From the ChlF records, the following ChlF parameters were evaluated: (i) Fv/Fm (maximum yield of PSII photochemical processes), (ii) Φ PSII (effective quantum yield of PSII) and (iii) relative decline of chlorophyll fluorescence (vitality index -Rfd). ...
Hydroponic experiments were performed to examine the effect of prolonged sulfate limitation combined with cadmium (Cd) exposure in Arabidopsis thaliana and a potential Cd hyperaccumulator, Nicotiana tabacum. Low sulfate treatments (20 and 40 µM MgSO4) and Cd stress (4 µM CdCl2) showed adverse effects on morphology, photosynthetic and biochemical parameters and the nutritional status of both species. For example, Cd stress decreased NO3⁻ root content under 20 µM MgSO4 to approximately 50% compared with respective controls. Interestingly, changes in many measured parameters, such as chlorophyll and carotenoid contents, the concentrations of anions, nutrients and Cd, induced by low sulfate supply, Cd exposure or a combination of both factors, were species-specific. Our data showed opposing effects of Cd exposure on Ca, Fe, Mn, Cu and Zn levels in roots of the studied plants. In A. thaliana, levels of glutathione, phytochelatins and glucosinolates demonstrated their distinct involvement in response to sub-optimal growth conditions and Cd stress. In shoot, the levels of phytochelatins and glucosinolates in the organic sulfur fraction were not dependent on sulfate supply under Cd stress. Altogether, our data showed both common and species-specific features of the complex plant response to prolonged sulfate deprivation and/or Cd exposure.
... For most tomato cultivars, if the temperatures exceed 26ºC during the day and 20ºC during the night, fruit set is impaired mainly due to floral development alteration, and therefore fruit yield is reduced (Sherzod et al., 2020). Because the response to stress varies widely between genotypes (Arena et al., 2020;Gonzalo et al., 2021), gaining deeper knowledge of the developmental and molecular response of various cultivars may provide clues for innovative breeding strategies aiming at improving commercial tomato cultivars for better adaptability to climate change. In the present work, we investigated the responses to heat stress in eleven tomato cultivars producing fruits of different sizes and shapes. ...
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Climate change is one of the biggest threats that human society currently needs to face. Heat waves associated with global warming negatively affect plant growth and development and will increase in frequency. Tomato is one of the most produced and consumed fruit in the world but remarkable yield losses occur every year due to the sensitivity of many cultivars to heat stress. New insights into how tomato plants are responding to heat waves will contribute to the development of new cultivars with high yields under harsh temperature conditions. In this study, the analysis of microsporogenesis and pollen germination rate of eleven tomato cultivars after exposure to a simulated heat wave revealed differences between genotypes. The transcriptome of floral buds at two developmental stages of five cultivars selected based on their pollen germination tolerance or sensitivity, revealed common and specific molecular responses implemented by tomato cultivars to cope with heat waves. These data provide valuable insights into the underlying molecular adaptation of floral buds to heat stress and will contribute to the development of future climate resilient tomato varieties.
Rising daily temperatures and water shortage are two of the major concerns in agriculture. In this work, we analysed the tolerance traits in a tomato line carrying a small region of the Solanum pennellii wild genome (IL12‐4‐SL) when grown under prolonged conditions of single and combined high temperature and water stress. When exposed to stress, IL12‐4‐SL showed higher heat tolerance than the cultivated line M82 at morphological, physiological, and biochemical levels. Moreover, under stress IL12‐4‐SL produced more flowers than M82, also characterized by higher pollen viability. In both lines, water stress negatively affected photosynthesis more than heat alone, whereas the combined stress did not further exacerbate the negative impacts of drought on this trait. Despite an observed decrease in carbon fixation, the quantum yield of PSII linear electron transport in IL12‐4‐SL was not affected by stress, thereby indicating that photochemical processes other than CO 2 fixation acted to maintain the electron chain in oxidized state and prevent photodamage. The ability of IL12‐4‐SL to tolerate abiotic stress was also related to the intrinsic ability of this line to accumulate ascorbic acid. The data collected in this study clearly indicate improved tolerance to single and combined abiotic stress for IL12‐4‐SL, making this line a promising one for cultivation in a climate scenario characterized by frequent and long‐lasting heatwaves and low rainfall.
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The Elongator complex in eukaryotes has conserved tRNA modification functions and contributes to various physiological processes such as transcriptional control, DNA replication and repair, and chromatin accessibility. ARABIDOPSIS ELONGATOR PROTEIN 4 (AtELP4) is one of the six subunits (AtELP1-AtELP6) in Arabidopsis Elongator. In addition, there is an Elongator-associated protein, DEFORMED ROOTS AND LEAVES 1 (DRL1), whose homolog in yeast (Kti12) binds tRNAs. In this study, we explored the functions of AtELP4 in plant-specific aspects such as leaf morphogenesis and evolutionarily conserved ones between yeast and Arabidopsis. ELP4 comparison between yeast and Arabidopsis revealed that plant ELP4 possesses not only a highly conserved P-loop ATPase domain but also unknown plant-specific motifs. ELP4 function is partially conserved between Arabidopsis and yeast in the growth sensitivity toward caffeine and elevated cultivation temperature. Either single Atelp4 or drl1-102 mutants and double Atelp4 drl1-102 mutants exhibited a reduction in cell proliferation and changed the adaxial-abaxial polarity of leaves. In addition, the single Atelp4 and double Atelp4 drl1-102 mutants showed remarkable downward curling at the whole part of leaf blades in contrast to wild-type leaf blades. Furthermore, our genetic study revealed that AtELP4 might epistatically act on DRL1 in the regulation of cell proliferation and dorsoventral polarity in leaves. Taken together, we suggest that AtELP4 as part of the plant Elongator complex may act upstream of a regulatory pathway for adaxial-abaxial polarity and cell proliferation during leaf development.
Tomato, an important vegetable crop cultivated worldwide is an essential component of the Ghanaian diet. However, production in Ghana is hampered by heat stress (HS) during certain periods of the year. Tomato is sensitive to HS, especially during the reproductive stage, inducing pollen sterility, floral and fruit abortion, and culminating in low yield. This necessitates development of HS-tolerant tomato varieties. The HS-tolerance potential of 13 tomato lines was assessed under both poly-house and open field conditions at day temperatures of ~35°C. Evaluation was based on agro-morphological and physiological traits. Most lines had their highest fruit yield (FY) under poly-house conditions, but declined drastically (up to 92%) in the field when day/night temperatures reached 40/28°C respectively. A second field experiment revealed that the low FY was due to poor fruit set despite the many flowers produced. Correlations between days to 50% fruiting and physiological traits (ΦII, chlorophyll content, and leaf temperature) were high and significant, whereas those between FY and all physiological parameters were negligible. Lines KAC13 and KAU03 consistently had high FY, ΦII, and chlorophyll content. Nevertheless, KAU01 had the highest FY in two out of three experiments in addition to having the highest percentage fruit set. The lines that show high potential tolerance to HS may be incorporated into breeding programmes to further improve the tolerance of tomatoes to HS.
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Abiotic stresses can cause a substantial decline in fruit quality due to negative impacts on plant growth, physiology and reproduction. The objective of this study was to verify if the use of a biostimulant based on plant and yeast extracts, rich in amino acids and that contains microelements (boron, zinc and manganese) can ensure good crop yield and quality in tomato plants grown at elevated temperatures (up to 42 °C). We investigated physiological responses of four different tomato landraces that were cultivated under plastic tunnel and treated with the biostimulant CycoFlow. The application of the biostimulant stimulated growth (plants up to 48.5% taller) and number of fruits (up to 105.3%). In plants treated with the biostimulant, antioxidants contents were higher compared to non-treated plants, both in leaves and in fruits. In particular, the content of ascorbic acid increased after treatments with CycoFlow. For almost all the traits studied, the effect of the biostimulant depended on the genotype it was applied on. Altogether, the use of the biostimulant on tomato plants led to better plant performances at elevated temperatures, that could be attributed also to a stronger antioxidant defence system, and to a better fruit nutritional quality.
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High temperature is one of the most detrimental abiotic stresses in tomatoes. Many studies highlighted that even small increases in temperature can alter the plant reproductive system, causing a significant reduction in tomato yield. The aim of this study was to exploit the phenotypic and genomic variations of a tomato landrace collection grown at high temperatures. Fifteen genotypes were selected as the best performing in two experimental fields. The selection was based on six yield-related traits, including flower earliness, number of flowers per inflorescence, fruit set, number of fruit per plant, fruit weight and yield per plant. In order to identify markers targeting traits that could be highly influenced by adverse climate conditions, such as flowering and fruit setting, an association mapping approach was undertaken exploiting a tomato high-throughput genomic array. The phenotypic variability observed allowed us to identify a total of 15 common markers associated with the studied traits. In particular, the most relevant associations co-localized with genes involved in the floral structure development, such as the style2.1 gene, or with genes directly involved in the response to abiotic stresses. These promising candidate genes will be functionally validated and transferred to a cultivated tomato to improve its performance under high temperatures
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Drought (water deficits) and heat (high temperatures) stress are the prime abiotic constraints, under the current and climate change scenario in future. Any further increase in the occurrence, and extremity of these stresses, either individually or in combination, would severely reduce the crop productivity and food security, globally. Although, they obstruct productivity at all crop growth stages, the extent of damage at reproductive phase of crop growth, mainly the seed filling phase, is critical and causes considerable yield losses. Drought and heat stress substantially affect the seed yields by reducing seed size and number, eventually affecting the commercial trait ‘100 seed weight’ and seed quality. Seed filling is influenced by various metabolic processes occurring in the leaves, especially production and translocation of photoassimilates, importing precursors for biosynthesis of seed reserves, minerals and other functional constituents. These processes are highly sensitive to drought and heat, due to involvement of array of diverse enzymes and transporters, located in the leaves and seeds. We highlight here the findings in various food crops showing how their seed composition is drastically impacted at various cellular levels due to drought and heat stresses, applied separately, or in combination. The combined stresses are extremely detrimental for seed yield and its quality, and thus need more attention. Understanding the precise target sites regulating seed filling events in leaves and seeds, and how they are affected by abiotic stresses, is imperative to enhance the seed quality. It is vital to know the physiological, biochemical and genetic mechanisms, which govern the various seed filling events under stress environments, to devise strategies to improve stress tolerance. Converging modern advances in physiology, biochemistry and biotechnology, especially the “omics” technologies might provide a strong impetus to research on this aspect. Such application, along with effective agronomic management system would pave the way in developing crop genotypes/varieties with improved productivity under drought and/or heat stresses.
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Elevated atmospheric CO2 improves leaf photosynthesis and plant tolerance to heat stress, however, the underlying mechanisms remain unclear. In this study, we exposed tomato plants to elevated CO2 (800 μmol mol-1) and/or high temperature (42∘C for 24 h), and examined a range of photosynthetic and chlorophyll fluorescence parameters as well as cellular redox state to better understand the response of photosystem II (PSII) and PSI to elevated CO2 and heat stress. The results showed that, while the heat stress drastically decreased the net photosynthetic rate (Pn), maximum carboxylation rate (Vcmax), maximum ribulose-1,5-bis-phosphate (RuBP) regeneration rate (Jmax) and maximal photochemical efficiency of PSII (Fv/Fm), the elevated CO2 improved those parameters under heat stress and at a 24 h recovery. Furthermore, the heat stress decreased the absorption flux, trapped energy flux, electron transport, energy dissipation per PSII cross section, while the elevated CO2 had the opposing effects that eventually decreased photoinhibition, damage to photosystems and reactive oxygen species accumulation. Similarly, the elevated CO2 helped the plants to maintain a reduced redox state as evidenced by the increased ratios of ASA:DHA and GSH:GSSG under heat stress and at recovery. Furthermore, the concentration of NADP+ and ratio of NADP+ to NADPH were induced by elevated CO2 at recovery. This study unraveled the crucial mechanisms of elevated CO2-mediated changes in energy fluxes, electron transport and redox homeostasis under heat stress, and shed new light on the responses of tomato plants to combined heat and elevated CO2.
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This study aimed to phenotype young tomato (Solanum lycopersicum L.) plants for heat tolerance by measuring Fv/Fm after short-term heat treatments in climate chambers and selected sensitive (low Fv/Fm) and tolerant (high Fv/Fm) cultivars to investigate their in-field performance. Twenty-eight genotypes were phenotyped at 40: 28°C for 2 days in climate chambers. A second screening (four high Fv/Fm and four low Fv/Fm genotypes) was conducted for 4 days at 38: 28°C, followed by 5 days' recovery (26: 20°C). The tolerant genotypes maintained high net photosynthesis (PN) and increased stomatal conductance (gs) at 38°C, allowing better leaf cooling. Sensitive genotypes had lower Fv/Fm and PN at 38°C, and gs increased less than in the tolerant group, reducing leaf cooling. Under controlled conditions, all eight genotypes had the same plant size and pollen viability, but after heat stress, plant size and pollen viability reduced dramatically in the sensitive group. Two tolerant and two sensitive genotypes were grown in the field during a heat wave (38: 26°C). Tolerant genotypes accumulated more biomass, had a lower heat injury index and higher fruit yield. To our knowledge, this is the first time screening for heat tolerance by Fv/Fm in climate chambers was verified by a field trial under natural heat stress. The differences after heat stress in controlled environments were comparable to those in yield between tolerant and sensitive groups under heat stress in the field. The results suggest that Fv/Fm is effective for early detection of heat tolerance, and screening seedlings for heat sensitivity can speed crop improvement.
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Despite a range of initiatives to reduce global carbon emission, the mean global temperature is increasing due to climate change. Since rising temperatures pose a serious threat of food insecurity, it is important to further explore important biological molecules that can confer thermotolerance to plants. Recently, melatonin has emerged as a universal abiotic stress regulator that can enhance plant tolerance to high temperature. Nonetheless, such regulatory roles of melatonin were unraveled mainly by assessing the effect of exogenous melatonin on plant tolerance to abiotic stress. Here, we generated melatonin deficient tomato plants by silencing of a melatonin biosynthetic gene, CAFFEIC ACID O-METHYLTRANSFERASE 1 (COMT1), to unveil the role of endogenous melatonin in photosynthesis under heat stress. We examined photosynthetic pigment content, leaf gas exchange, and a range of chlorophyll fluorescence parameters. The results showed that silencing of COMT1 aggravated heat stress by inhibiting both the light reactions and the carbon fixation reactions of photosynthesis. The photosynthetic pigment content, light absorption flux, trapped energy flux, energy dissipation, density of active reaction center per photosystem II (PSII) cross-section, the photosynthetic electron transport rate, the maximum photochemical efficiency of PSII photochemistry, and the rate of CO2 assimilation all decreased in COMT1-silenced plants compared with that of non-silenced plants particularly under heat stress. However, exogenous melatonin alleviated heat-induced photosynthetic inhibition in both genotypes, indicating that melatonin is essential for maintaining photosynthetic capacity under stressful conditions. These findings provide genetic evidence on the vital role of melatonin in photosynthesis and thus may have useful implication in horticultural crop management in the face of climate change.
Mediterranean tomato landraces adapted to arid environments represent an option to counteract drought, and to address the complexity of responses to water deficit and recovery, which is a crucial component of plant adaptation mechanisms. We investigated physiological, biochemical and molecular responses of two Mediterranean tomato landraces, “Locale di Salina” (Lc) and “Pizzutello di Sciacca” (Pz) under two dehydration periods and intermediate rehydration in greenhouse pot‐experiments. Relationship between CO2 assimilation and stomatal conductance under severe water stress (gs < 0.05 mol m⁻² s⁻¹) indicated the occurrence of stomatal and non‐stomatal limitations of photosynthesis. Gas‐exchanges promptly recovered within 2‐3‐day re‐hydration. ABA and gs showed a strict exponential relationship. Both leaf ABA and proline peaked under severe water stress. Lc showed higher accumulation of ABA and higher induction of the expression of both NCED and P5CS genes than Pz. PARP increased during imposition of stress, mainly in Lc, and decreased under severe water stress. The two landraces hardly differed in their physiological performance. Under severe water stress, stomatal conductance showed low sensitivity to ABA, which instead controlled stomatal closure under moderate water stress (gs > 0.15 mol m⁻² s⁻¹). The prompt recovery after re‐dehydration of both landraces confirmed their drought tolerant behaviour. Differences between the two landraces were instead observed at biochemical and molecular levels. This article is protected by copyright. All rights reserved.
Tomato cultivation at lower or higher temperatures than the optimum negatively affects plant growth and development. Large differences in abiotic stress tolerance have been found between Solanum lycopersicum and wild tomato species. Our aim was to compare temperature stress tolerance in cultivated and wild tomato genotypes to identify cold- and heat-tolerant tomatoes for further utilization in tomato breeding. The maintained net photosynthetic rate (PN) and chlorophyll fluorescence was related to the tolerance of tomatoes at temperature stress. The PN and chlorophyll fluorescence of one cultivated tomato (Ly from S. lycopersicum) and six wild tomatoes genotypes (Ha from Solanum habrochaites, Pe from Solanum pennellii, Pi1 and Pi2 from Solanum pimpinellifolium, Pr1 and Pr2 from Solanum peruvianum) grown at low (12 °C) and high (33 °C) temperatures were compared. The PN of four tomato genotypes during temperature stress were lower than the control, but not in Pe, Pr1, and Pr2. The maximum quantum efficiency of photosystem II (Fv/Fm) of the cultivated tomatoes was lower at both 12 and 33 °C than the control using Handy PEA, whereas Fv/Fm using MINI-PAM was lower only at 12 °C. The chlorophyll fluorescence OJIP transient (OJIP curve) revealed differences between temperature stress responses and tomato genotype. With the exception of Pr2, the Fv/Fm in wild tomatoes was unaffected by temperature stress; however, they still maintained clear genotype differences for other physiological traits such as PN, quantum yield of PSII (Fq′/Fm′), electron transport rate, non-photochemical quenching, and the fraction of open PSII centers (qL). These results indicated that the wild tomato varieties Pe and Pr1 had the highest temperature stress tolerance, while the cultivated species was the more sensitive to temperature stress in comparison. In general, the wild tomato genotypes were more tolerant to both cold and heat stress than the cultivated tomato, suggesting that these wild species could be used to uncover underlying mechanisms of temperature stress tolerance and will be promising sources of genetic variability for temperature stress tolerance in breeding programs.