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Abstract
Temperature is one of the main environmental factors involved in global warming and has been found to have a direct effect
on plants. However, few studies have investigated the effect of higher temperature on tropical crops. We therefore
performed an experiment with a tropical crop of Habanero pepper (Capsicum Chinense Jacq.). Three growth chambers
were used, each with 30 Habanero pepper plants. Chambers were maintained at a diurnal maximum air temperature (DMT)
of 30 (chamber 1), 35 (chamber 2) and 40uC (chamber 3). Each contained plants from seedling to fruiting stage.
Physiological response to variation in DMT was evaluated for each stage over the course of five months. The results
showed that both leaf area and dry mass of Habanero pepper plants did not exhibit significant differences in juvenile and
flowering phenophases. However, in the fruiting stage, the leaf area and dry mass of plants grown at 40 uC DMT were 51
and 58% lower than plants at 30uC DMT respectively. Meanwhile, an increase in diurnal air temperature raised both
stomatal conductance and transpiration rate, causing an increase in temperature deficit (air temperature leaf
temperature). Thus, leaf temperature decreased by 5uC, allowing a higher CO2 assimilation rate in plants at diurnal
maximum air temperature (40uC). However, in CO2 measurements when leaf temperature was set at 40uC, physiological
parameters decreased due to an increase in stomatal limitation. We conclude that the thermal optimum range in a tropical
crop such as Habanero pepper is between 30 and 35uC (leaf temperature, not air temperature). In this range, gas
exchange through stomata is probably optimal. Also, the air temperatureleaf temperature relationship helps to explain how
temperature keeps the major physiological processes of Habanero pepper healthy under experimental conditions.
Citation: Garruna-Hernandez R, Orellana R, Larque-Saavedra A, Canto A (2014) Understanding the Physiological Responses of a Tropical Crop (Capsicum chinense
Jacq.) at High Temperature. PLoS ONE 9(11): e111402. doi:10.1371/journal.pone.0111402
Editor: Marcel Quint, Leibniz Institute of Plant Biochemistry, Germany
Received January 8, 2014; Accepted October 2, 2014; Published November 3, 2014
Copyright: 2014 Garruna-Hernandez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The Mexican Council of Science and Technology (CONACYT) supports the doctoral studies of Rene Garruna-Hernandez. The funders had
no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* Email: renegh10@hotmail.com
physiological traits in Habanero pepper plants. Three specific samples were dried in an oven at 65uC until constant weight was
questions are addressed here: What are the physiological reached and then dry mass (DM) was determined. Relative
responses of C. chinense to air temperature increase? What is growth rate (RGR), leaf area ratio (LAR), specific leaf area
the thermal optimum of this tropical crop under changing (SLA), net assimilation rate (NAR) and leaf weight ratio (LWR)
environmental conditions? What physiological strategies are were calculated as described by Hunt et al. [14]. These five
involved in achieving physiological plasticity? parameters are defined and related in the following way:
Experimental design The dry mass proportion per organ (DM%) is an indicator of
A completely random design was used with 30 plants per how temperature affects each plant organ. In this case, dry mass
growth chamber. Data were examined by ANOVA and treatment proportion of Habanero pepper plants changed in each phenology
means were compared using Tukeys HSD test at P,0.05 (Statistic stage. However, DM% of roots, stems, leaves and flowers did not
Six Sigma, Release 7, StatSoft). An arcsine square root transfor- exhibit significant differences between treatments. But, dry mass
mation was used to adjust the normality of the data percentages proportion in fruiting at 30uC was at least 37% higher than others
(in dry mass proportion). treatments (Figure 2).
Growth parameters show the use of resources by the plant. In
Results all treatments, relative growth rate based on dry mass (RGR DW),
relative growth rate based on leaf area (RGR LA) and net
High temperature affects plant growth. In this study, both leaf assimilation rate (NAR) did not differ during flowering and
area and dry mass of Habanero pepper plants did not exhibit fruiting stages. However, in juvenile plants at 40uC DMT, these
significant differences in juvenile and flowering phenophases. three parameters were 37, 44 and 37% higher than plants at 30uC
However, in the fruiting phenophase, the leaf area and dry mass DMT respectively (Figures 3A, 3B and 3C). Leaf area ratio
of plants grown at 40uC DMT were 51 and 58% lower than plants (LAR) and the specific leaf area (SLA) did not exhibit significant
at 30uC DMT respectively (Figures 1A and 1B). differences in juveniles, or in flowering and fruiting stages. Both
were significantly reduced by 32 and 34% respectively when
Figure 1. Leaf area and dry mass of Habanero pepper plants. Figure 1. A) Leaf area and B) dry mass of Habanero pepper plants in different
phenological stages at three maximum diurnal temperatures (T air = 30, 35 and 40uC). Data are means 6 SE. Different letters in the same
phenology stage represent statistically significant differences (Tukey, a = 0.05). n = 5.
doi:10.1371/journal.pone.0111402.g001
Figure 2. Dry mass proportion in different phenophases. Figure 2. Average dry mass proportion of Habanero pepper plants in different
phenological stages at three maximum diurnal temperatures (Tair = , 35 and 40uC). Different letters in the same organ represent statistically
significant differences (Tukey, a = 0.05). n = 5.
doi:10.1371/journal.pone.0111402.g002
temperature was increased from 30 to 40uC during seedling 35 and 40uC DMT, the leaf temperature was ca. 27.8, 30.5 and
stages (Figures 3D and 3E). In contrast, leaf weight ratio (LWR) 35uC, respectively. This result was significant in all treatments
was not significant in either seedling or juvenile stages, but (Figure 5F).
increased by 23 and 44%, respectively in flowering and fruiting In the ACi curves, the photosynthetic system of leaves is
stages when temperature was increased from 30 to 35uC (Figure gradually saturated with CO2, providing a measure of physiolog-
3F). Probably the results of juvenile plants were a consequence of ical plasticity of the plant in different environments. The ACi
few days within the chamber. curve analyses conducted on Habanero pepper plant leaves
Stomata permit gas exchange between the inside and outside of showed that at a leaf temperature of 40uC (not DMT), there was a
plants. An increase in stomata number could change the gas-exchange decrease in the maximum photosynthetic activity (52%), intercel-
rate. The occurrence of amphistomatous leaves is not unusual for lular CO2 (15%) and stomatal conductance (50%) as a result of a
pepper plants, although it has not been reported for this species. 33% increase of in stomatal limitation (Figure 6).
Therefore, amphistomatous leaves are reported in this study on
Habanero pepper plants. However, in juvenile, flowering and fruiting Discussion
stages the stomatal number in the adaxial side was at least 81, 95 and
87% lower than on the abaxial side respectively. The stomatal density An increase in maximum temperature above the thermal optimum
on abaxial sides decreased from juvenile to flowering stage. should have a negative effect on physiological perfor-mance. For
Additionally, stomatal number decreased from the upper leaf section example, photosynthesis [17], [18], respiration [19], plant growth
to the lower leaf section (Figure 4). [14] and reproductive processes such as flowering and fruit set may
Physiological parameters are fundamental for understanding be especially sensitive to high temperatures [20],
plants responses to the environment. In this study, plants at 40uC [21]. However, the thermal optimum is higher in a tropical plant
DMT had a net CO2 assimilation rate that was 20 and 57% higher than in a temperate plant, because tropical plants are high
than those at 30uC DMT at 12:00 and 14:30 h respectively temperature adapted species [22].
(Figure 5A), when photosynthetic photon flux density was at its According to our results, the thermal optimum for leaf
maximum (PPFD). In addition, stomatal conductance at 40uC temperature in Habanero pepper should be below 35uC
DMT was 57% higher than in plants at 30uC DMT at 12:00 h (Figure 16). Although the decrease in leaf temperature should be
(Figure 5B). Intercellular CO2 (Ci) in plants at 40uC DMT was between 1 or 2uC with respect to air temperature [23], according
higher than in plants at 30 and 35uC DMT between 9:00 and to our results, a rise in diurnal maximum temperature increased
14:30 h (Figure 5C). At noon, there was no difference in the the transpiration rate (Figure 5E) with the effect that this
intercellular CO2/atmospheric CO2 ratio between treatments, but kept leaf temperatures close to the thermal optimum range for
at 7:00 and 17:00 h the plants measured in all treatments were Habanero pepper (between 3035uC). This caused the tempera-
close to 1 (Figure 5D). Transpiration rate increased in higher ture to decrease to 5uC below the air temperature (Figure 5F).
temperature treatments (35 and 40uC DMT) during the course of Thus, we consider that the Tair Tleaf relationship describes the
the day (Figure 5E) and was very similar to stomatal conductance. maintenance of healthy major physiological processes (i.e.
Temperature deficit (Tair Tleaf) in plants at 40uC reached 5uC at photosynthesis), causing a rise in both stomatal conductance
12:00 h and 4.6uC at 14:30 h; ca. 2.7 and 3.3uC higher than (Figure 5B) and intercellular CO2 concentration (Figure 5C), as
plants at 30uC. Although air temperature in each chamber was 30,
Figure 3. Growth parameters of Habanero pepper plants. Figure 3. A) Relative growth rate (dry mass); B) Relative growth rate (leaf area);
C) Net assimilation rate; D) Leaf area ratio; E) Specific leaf area; and F) Leaf weight ratio of Habanero pepper plants at three maximum
diurnal temperatures (Tair = 30uC, 35uC and 40uC) by phenological stage. Data are means 6 SE. Different letters in the same phenology
stage represent statistically significant differences (Tukey, a = 0.05). n = 5.
doi:10.1371/journal.pone.0111402.g003
well as a higher CO2 assimilation rate than observed at low and fruits (Figure 2). We observed that DMT Habanero pepper
temperatures (Figure 5A). plants at 40uC decreased their fruit set three-fold with respect to
Inhibition of photosynthesis by heat stress commonly occurs in values obtained for plants at 30uC DMT [25]. Moreover, flower
tropical and subtropical plants [22]. However, the optimal abortion of plants at 40uC DMT increased two-fold when
temperature for photosynthesis varies to a limited extent compared to plants at 30uC DMT, probably due to hormonal
depending upon the conditions for growth [24]. Furthermore, imbalance as a consequence of elevated temperature [25].
some growth parameters such as leaf area (Figure 1A) and dry Hormonal activity has an effect on abortion of flowers and fruits
mass (Figure 1B) exhibited lower values at higher diurnal in reproductive phenophases [26]. This suggests that an elevated
temperature in reproductive processes (flowering and fruiting). diurnal maximum air temperature does not have a negative effect
The latter was attributed to an increase in abortion of both flowers on the photosynthetic apparatus in a tropical plant like Habanero
Figure 4. Stomata number of Habanero pepper plants. Figure 4. Stomata number (adaxial and abaxial) at three height levels of plants (top,
middle, and bottom) and different phenological stages, A) Adaxial juvenile; B) Adaxial flowering; C) Adaxial fruiting; D) Abaxial juvenile; E) Abaxial
flowering and F) Abaxial fruiting) of Habanero pepper plants at three maximum diurnal temperatures (T air = 30, 35 and 40uC). Data are means 6 SE.
Different letters represent statistically significant differences (Tukey, a = 0.05). n = 5. The scale of graphs on the left is different to the graphs on the
right.
doi:10.1371/journal.pone.0111402.g004
pepper (Figure 5A), but could have a negative effect on hormonal The purpose of taking data measurements over the whole day
activity and diminish some major economic traits in tropical was to identify at what point the CO2 assimilation rate decreases
crops. However, if air temperature is very high (above 40uC) and as the air temperature rises. About 30% of the carbohydrate
the plants regulatory mechanism could not maintain the leaf formed in C3 photosynthesis is lost through photorespiration [27]
temperature at its thermal optimum, then a decrease in and the loss increases with temperature. However, in Habanero
intercellular CO2 concentration, maximum photosynthetic rate pepper under the established temperature intervals, CO 2 assim-
and stomatal conductance (ge) would be caused by an increase in ilation rate was always observed during diurnal measurements
stomatal limitation, as was observed in the results (Figure 6). (Figure 5A).
Figure 5. Physiological responses to increased air temperature. Figure 5. A) CO 2 Assimilation rate (AN); B) Stomatal conductance (ge); C)
Intercellular CO2 concentration (Ci); D) Ratio of intercellular CO2/atmospheric CO2 (CI/Ca); E) Transpiration (E); and F) Temperature deficit
(Tair Tleaf) of Habanero pepper plants at three maximum diurnal temperatures (T air = 30uC, 35uC and 40uC). Data are means 6 SE. n = 15.
NS: no significance, *: significant (ANOVA, P,0.05).
doi:10.1371/journal.pone.0111402.g005
Figure 6. Physiological responses to increased leaf temperature. Figure 6. Intercellular CO 2 concentration (Ci), maximum photosynthetic rate
(Amax), stomatal conductance (ge) and stomatal limitation (l) of Habanero pepper plants at three leaf temperatures (Tleaf = 30uC, 35uC and
40uC). Data are means 6 SE. Different letters in the same parameter represent statistically significant differences (Tukey, a = 0.05). n = 5.
doi:10.1371/journal.pone.0111402.g006
assimilation rate in plants at diurnal maximum air temperature of producing biomass. In consequence, the physiological process in
(40uC). However, in ACi curves when leaf temperature was set plants is maintained, avoiding damage due to high temperatures.
at 40uC, physiological parameters decreased due to an increase in
stomatal limitation. We showed that the thermal optimum range in Acknowledgments
a tropical crop such as Habanero pepper is between 30 and 35uC
We thank Celene M. Espadas, Lilia E. Carrillo and Rosalina Rodriguez
(leaf temperature, not air temperature). In this range, gas for comments and discussions on the manuscript. Thank you to Roberth
exchange through stomata is probably optimal in tropical species Us, Luis Sima and Isaac Castillo for their assistance in the field.
for good performance. Unexpectedly, CO 2 assimilation rate was
always present in our experiments. Although an increase in air Author Contributions
temperature enhanced photosynthesis values at diurnal measure-
Conceived and designed the experiments: RGH RO AC. Performed the
ments, this was not reflected in plant production (leaf area, dry
experiments: RGH RO AC. Analyzed the data: RGH RO AC ALS.
mass and relative growth rates). Thus, photosynthate surplus is Contributed reagents/materials/analysis tools: RGH RO AC ALS. Wrote
probably channelled and used in other metabolic pathways instead the paper: RGH RO AC.
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