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The response of contrasting tomato genotypes to combined heat and drought stress
Accepted Manuscript Title: The response of contrasting tomato genotypes to combined heat and drought stress Author: Alliea Nankishore Aidan D. Farrell PII: DOI: Reference:
S0176-1617(16)30126-2 http://dx.doi.org/doi:10.1016/j.jplph.2016.07.006 JPLPH 52405
To appear in: Received date: Revised date: Accepted date:
6-5-2016 6-7-2016 6-7-2016
Please cite this article as: Nankishore Alliea, Farrell Aidan D.The response of contrasting tomato genotypes to combined heat and drought stress.Journal of Plant Physiology http://dx.doi.org/10.1016/j.jplph.2016.07.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The response of contrasting tomato genotypes to combined heat and drought stress Alliea Nankishorea and Aidan D Farrellb* a
Department of Biology, University of Guyana, Guyana
b
Department of Life Sciences, University of the West Indies, St. Augustine Campus, Trinidad
and Tobago W.I.
Corresponding Author: * Aidan Farrell [email protected]
The response of contrasting tomato genotypes to combined heat and drought stress
ABSTRACT Efforts to maximize yields of food crops can be undermined by abiotic stress factors, particularly those related to climate change. Here, we use a range of physiological methods to detect the individual and combined effects of heat and drought stress on three contrasting varieties of tomato: Hybrid 61, Moskvich, and Nagcarlang. Seedlings were acclimated under the following treatment regimes: CONTROL (25-36 ⁰C; well-watered), DRY (25-36 ⁰C; 20 % field capacity), HOT (25-42 ⁰C; well-watered) and HOT+DRY (25-42 ⁰C; 20 % field capacity). In each treatment, stomatal conductance, leaf temperature, chlorophyll content, and several chlorophyll fluorescence variables (both in situ and in vitro following a heat shock treatment) were measured. Plants from the HOT treatment remained statistically similar to the CONTROL in all the measured parameters, while those from the DRY treatment and especially the HOT+DRY treatment showed clear effects of abiotic stress. Hybrid 61 showed considerable resilience to heat and drought stress compared to the other varieties, with significantly cooler leaves (one day after treatments imposed) and significantly higher Fv/Fm values both in situ and in vitro. The genotypic differences in resilience to heat stress were only apparent under water-limited conditions, highlighting the need to consider leaf temperature rather than air temperature when testing for tolerance to heat stress. The most effective parameters for discriminating genotypic variation in heat and drought stress were in vitro Fv/Fm and chlorophyll content.
Keywords: climate change; heat stress; water stress; plant breeding; leaf temperature; chlorophyll fluorescence.
INTRODUCTION
The global human population is currently growing at an unprecedented rate and is expected to remain on this trajectory for at least 35 years (Zargar et al., 2011; Meeks et al., 2013). An increasing population is associated with an increase in demand for food and this is projected to continue until food production has doubled (Howden et al., 2007; Bita and Gerats, 2013). However, efforts to grow and maximize yields of food crops can be undermined by climatic changes, such as increases in atmospheric temperature and decreases in precipitation (Zargar et al., 2011; Meeks et al., 2013; Eitzinger et al., 2015a, 2015b). Changes in these abiotic factors may induce physiological stress such as heat stress and drought stress in agronomically important plants. Drought stress is considered to be the most damaging abiotic stress to crop productivity (Foolad et al., 2003; Mir et al., 2012). High temperatures can also impact on crop productivity both directly and by exacerbating the effects of drought by promoting evapotranspiration (Farrell, 2014; Webber et al., 2015; Feller, 2016). These are major challenges in rainfed agriculture, especially in the arid and semi-arid regions of the tropics (Tomar and Kumar, 2004; Kulkarni and Deshpande, 2007). It is, therefore, vital to be able to identify and develop crop varieties that are resilient to abiotic stress so that crop productivity is not unduly affected (Foolad et al., 2003; Camejo et al., 2005; Bita et al., 2011; Feller and Vaseva, 2014). Tomato (Solanum lycopersicum L.) grows optimally at temperatures ranging from 20-30 ⁰C and is sensitive to extreme temperatures (Zhou et al., 2015) as well as water deficits (Petrozza et al., 2014). Tomato cultivation has increased in the tropics and subtropics where there is considerable risk from high temperature and drought periods. Previous studies have
demonstrated the drastic impacts of heat on tomato physiology (Sato et al., 2000; Singh et al., 2005; Camejo et al., 2006). Measures of stomatal conductance and leaf surface temperature are useful in determining the effects of stress on plant water relations and on their ability to avoid overheating, while measurements of chlorophyll content and chlorophyll fluorescence help to assess the level of stress-induced damage to photosynthetic structures and so indicate heat tolerance (Wahid et al., 2007; Farrell, 2014; Feller, 2016). Camejo et al. (2006) compared the response of heat-tolerant (Nagcarlang) and heat-sensitive (Amalia) varieties of tomato to heat stress after exposing seedlings to a heat shock at 45 ⁰C for 3 hours. Severe reductions in photosynthesis, stomatal conductance and chlorophyll content were observed in Amalia while Nagcarlang was unaffected. Recently, Zhou et al. (2015) observed similar reductions in these parameters in two heat-sensitive tomato varieties, relative to two heat-tolerant varieties. These same physiological parameters are seen to be impacted when tomato is grown under water deficit (Hayat et al., 2008; Zhang et al., 2011) although, in some cases, stomatal conductance and chlorophyll content have been seen to increase under heat stress while they typically decrease under drought stress (Zhou et al., 2015; Feller, 2016). Thus, there is a need to understand how the interaction between these physiological parameters contribute to genotypic variation in stress tolerance. Here, we use both in situ and in vitro physiological parameters measured in control and stress-acclimated tissue. In vitro stress testing provides a convenient technique to make uniform comparisons between varieties. In the case of high temperature stress, many studies have used an in vitro heat-shock method to estimate the overall thermal tolerance of leaf tissue (Willits and Peet, 2001; Camejo et al., 2005; Camejo et al., 2006). Exposing harvested leaf tissues to high temperatures in vitro for a relatively short period of time allows for identification of the lethal
temperature, above which there is disruption of metabolic processes and irreversible injury. Prior in situ acclimation to abiotic stress is an essential step, as it helps to enhance the ability of tissue to withstand future exposure to heat shock conditions (Mittler et al., 2011; Farrell, 2014). Mittler (2006) and Feller (2016) highlight the need to develop crops with enhanced resilience to a combination of different stresses and point to the potential negative and complex interaction between drought and high temperature. Drought-tolerant plants may not necessarily be tolerant to heat stress, and vice versa (Jagadish et al., 2011; Feller, 2016). Hence, we aim to examine the response to heat and/or drought in seedlings of three contrasting tomato varieties. Seedling response is evaluated using stomatal conductance, leaf surface temperature, chlorophyll content and chlorophyll fluorescence (measured in situ and in vitro). In particular, we seek to understand the role of heat avoidance, heat acclimation and heat tolerance processes in enabling resilient varieties to withstand the combined effects of heat and drought as experienced by plants under field conditions.
MATERIALS AND METHODS
Planting Materials The varieties used in our study were Nagcarlang, Hybrid 61 and Moskvich. Nagcarlang is heattolerant and is a wildtype, heirloom variety that originated in the Philippines (Camejo et al., 2005). Hybrid 61 is commonly grown in tropical regions and is known to be high-yielding and tolerant to harsh weather conditions, although it is little studied (Ali et al., 2015). In contrast,
Moskvich is sensitive to heat and is another heirloom variety with origins in Russia (Kamel et al., 2010). Tomato seeds were germinated at 22 ⁰C in the laboratory and then transplanted to 16ounce polystyrene cups with drainage holes, containing a 2:1 mixture of sharp sand and ProMix (Premier Tech Horticultural Inc., Quakertown, PA, USA). The plants were reared in a wellventilated, full-sun greenhouse at The University of the West Indies in St. Augustine, Trinidad, and were watered daily to field capacity. The daily photoperiod was approximately 12 hours and the typical mid-day sunlight within the greenhouse was 600 µmol/m2/s.
Acclimation At the 4-leaf stage, the varieties were arranged in a complete randomized block design, with 10 replicates per block and four treatments (CONTROL, HOT, DRY and HOT+DRY). Data loggers (HOBO Pro, Onset Corp., USA) were placed on each bench at canopy height to monitor air temperature and humidity. Each treatment was applied on adjacent benches as follows: 1. CONTROL: served as the control and were watered once a day with 150 mL of water. 2. HOT: bench was covered in a clear polythene sheet at a height of 1 m, which reduced the ventilation but allowed limited airflow from beneath the bench. Plants were watered once a day. 3. DRY: plants were partially deprived of water. Each pot received 20 mL of water per day, to maintain soil moisture at approximately 20 % of the soil field capacity. Soil volumetric water content was monitored 3 times per week from 6 replicates of each variety prior to rewatering, using a soil moisture meter (Fieldscout TDR 100 Soil Moisture Meter; Spectrum Technologies, Inc., Illinois).
4. HOT+DRY: HOT and DRY treatments were combined.
Over the course of the experiment on the uncovered benches (CONTROL and DRY), the mean daily air temperature was 29 ⁰C, with a mean daily range of 25-36 ⁰C, the mean daily relative humidity was 75 % with a mean daily range of 39-100 %, and the mean daily vapour pressure deficit was 0.9 kPa with a daily range of 0-2.9 kPa (HOBO Pro, Onset Corp., USA). On the covered benches (HOT and HOT + DRY) the mean daily air temperature was 30 ⁰C (range 25-42 ⁰C), the mean daily relative humidity was 73 % (range 30-100 %) and the mean daily vapour pressure deficit was 1.3 kPa (range 0-4.4 kPa). The different values on the covered benches were due to elevated mid-day temperatures, which were on average 5 ⁰C hotter than the uncovered benches. During and after acclimation, several physiological parameters were measured as set out in Table 1. All measurements were made mid-way along the leaf, using the first fully expanded leaf. Stomatal conductance was measured immediately after taking leaf temperature. The thermal camera was held at an angle of 45⁰ from the leaf, at a distance of approximately 0.45 m and at an emissivity of 0.98. Chlorophyll content was measured on a per area basis as the ratio of light absorbsion at 660 nm to that at 940 nm (SPAD equivalent ratio units; Zhu et al., 2012).
Table 1 Outline of the timing of all measurements Measurement
Instrument Used
Stomatal conductance
Leaf porometer (SC-1, Decagon Devices; Pullman; WA, USA)
Days from Treatment Time of Measurement 1 2 3 4 5 6 7 8 9 10 11 12 14 Twice weekly during mid-day hours (10:0014:00 h)
Leaf surface temperature
Thermal camera (TI400, Fluke, USA)
Twice weekly during mid-day hours
Chlorophyll content
Chlorophyll meter (atLEAF+, FT Green LLC, Detroit, USA)
Once weekly after midday hours
Dark adapted chlorophyll fluorescence
Pulse modulated chlorophyll fluorometer (Mini-PAM; Walz; Effeltrich, Germany)
Once weekly at least 1 hour after dark (20:0021:30 h)
Light adapted chlorophyll fluorescence
Pulse modulated chlorophyll fluorometer (Mini-PAM; Walz; Effeltrich, Germany)
Once weekly during mid-day hours
In vitro chlorophyll fluorescence
Pulse modulated chlorophyll fluorometer (Mini-PAM; Walz; Effeltrich, Germany)
Once post-acclimation
A modulated chlorophyll fluorometer was used to estimate the steady state quantum yield of photosystem II in the light (at mid-day; Fq’/Fm’) and the maximum quantum yield of photosystem II following dark-adaptation (one hour after nightfall; Fv/Fm) (Baker, 2008).
Laboratory Measurements After acclimation, the plants were transported from the greenhouse to the laboratory and placed in a dark room for 40 minutes in order to dark adapt. One leaf was harvested from each plant and rinsed in 20 mL distilled water. In vitro Fv/Fm was obtained for each leaf using a pulse modulated chlorophyll fluorometer (Mini-PAM; Walz; Effeltrich, Germany). Each leaf was then placed in a labeled heat-shock vial containing 20 mL de-ionized water and left in a preheated (43 ⁰C) water bath for 1 hour, after which fluorescence measurements were repeated. The difference between measurements taken before and after the heat-shock were expressed as a percentage: (fluorescence after heat treatment / fluorescence before heat treatment × 100). A pilot study with
control plants showed that leaves exposed to a room temperature (26 ⁰C) treatment were unaffected by the in vitro incubation.
Statistical Analysis Data were analyzed using the statistical software R (Version 3.2.1, R Foundation for Statistical Computing, Vienna, Austria) for two-way analysis of variance (ANOVA) and post-hoc Fisher’s Least Significant Difference (LSD). Plants were grouped with treatment and variety as factors. Differences were considered as significant when p < 0.05.
RESULTS
Stomatal conductance Stomatal conductance after 1 day of exposure to heat and/or drought stress (Fig. 1A) differed significantly among treatments (p < 0.001) and among varieties (p < 0.05), but the interaction between these two factors was not significant (p > 0.05). Plants under the HOT treatment had elevated stomatal conductance, although the differences were not significant for individual varieties. Conductance readings for plants from the DRY treatment were lower than those from the CONTROL treatment, while those from the HOT+DRY treatment were significantly lower than the HOT treatment. Of the three varieties, Hybrid 61 showed the highest conductance across all treatments.
Fig. 1. Stomatal conductance (SC; mmol m-2s-1) and leaf temperature (LT; ⁰C) after 2 days (A and C, respectively) and 9 days (B and D, respectively) of stress. Bars represent means + standard errors, n = 10. Bars that do not have a common letter are significantly different within each sub-figure according to Fisher’s LSD test.
On day 9, plants from the CONTROL and the HOT treatments showed higher conductance levels than seen on the previous measurement day while those from the DRY and the HOT+DRY treatments were lower (Fig. 1B). There were significant differences between treatments (p < 0.001) but not between the other factors. Conductance readings for plants from the DRY treatment were significantly lower than those from the CONTROL treatment, while those from the HOT+DRY treatment were significantly lower than the HOT treatment. Differences between treatments were more pronounced than on day 2, but genotypic differences were less apparent.
Leaf surface temperature After 1 day of exposure to heat and/or drought stress, plants showed significant differences among treatments (p < 0.001) and near significant differences among varieties (p = 0.058). The interaction between these two factors was not significant. Plants from the CONTROL and the HOT treatments had similar leaf temperatures (Fig. 1C) but these were significantly lower than those from the DRY and the HOT+DRY treatments, which were also similar to each other. In the HOT+DRY, Hybrid 61, which had the highest stomatal conductance, also had the lowest leaf temperature (significantly lower than Nagcarlang). On day 9, differences in leaf surface temperature among treatments remained significant (p < 0.001), while differences among varieties and for the interaction were not significant. Plants from the CONTROL and the HOT treatments still maintained similar leaf temperatures. Plants in the DRY treatment, with negligible stomatal conductance, remained significantly hotter than those in the CONTROL treatment, while plants in the HOT+DRY treatment were significantly hotter than all other treatments (Fig. 1D).
Chlorophyll content After 10 days of exposure to the stress conditions, the chlorophyll content of leaves showed significant differences among treatments (p < 0.001) and varieties (p < 0.05), but no significant interaction. For all three varieties, plants in the DRY treatment had less chlorophyll than those in the CONTROL treatment (not significant). In the HOT+DRY treatment, Nagcarlang and Moskvich had lower values than in the other treatments, while Hybrid 61 maintained its chlorophyll content in all treatments (Fig. 2).
Fig. 2. Chlorophyll content (ratio units) after 10 days of stress. Bars represent means + standard errors, n = 10. Bars that do not have a common letter are significantly different according to Fisher’s LSD test.
In situ chlorophyll fluorescence After 10 days of exposure to heat and/or drought stress, Fo was significantly different among varieties (p < 0.001) but not among treatments or in the interaction (Fig. 3A). Fm was significantly different among treatments (p < 0.001), with lower values seen under DRY and HOT+DRY conditions, but not among varieties or in the interaction (Fig. 3B). Fv/Fo was significantly different among treatments (p < 0.05) and varieties (p < 0.001), but not in the interaction, while Fv/Fm was significant for all factors. For Nagcarlang and Moskvich, plants in
the DRY treatment had lower Fv/Fo and Fv/Fm values than in the CONTROL treatment (significantly lower for Moskvich), while Hybrid 61 maintained high values across all treatments. The HOT+DRY treatment also showed lower Fv/Fo and Fv/Fm values for Moskvich compared to CONTROL and HOT plants, although these were not lower than the values from the DRY treatment (Figs. 3C and 3D). Fq’/Fm’ was significantly different among treatments (p < 0.001), but not among varieties or in the interaction. Plants from the DRY and HOT+DRY treatments were significantly lower than the CONTROL but similar to each other (Fig. 3F). No genotypic differences were apparent.
Fig. 3. Sub-figures A, B, C, D and F represent dark-adapted mean Fo, Fm, Fv/Fo, Fv/Fm and light adapted mean Fq’/Fm’, respectively, after 10 days of stress, while sub-figure E illustrates mean difference in Fv/Fm after in vitro heat-shock. Bars represent means + standard errors, n =
10. Bars that do not have a common letter are significantly different within each sub-figure according to Fisher’s LSD test.
In vitro chlorophyll fluorescence Following exposure to 43 ⁰C for 1 hour, in vitro fluorescence varied significantly among treatments (p < 0.001), but not among varieties or in the interaction between these two factors. Plants from the HOT and the DRY treatments tended to withstand the heat-shock better than plants from the CONTROL (only significant for Nagcarlang). Plants from the HOT+DRY treatment maintained significantly higher post heat-shock Fv/Fm values when compared to all other treatments (Fig. 3E). Hot acclimated Hybrid 61 plants were significantly more tolerant to heat shock than Moskvich.
DISCUSSION
Our treatments included relatively brief periods of very high temperatures occurring at mid-day, similar to conditions experienced under cultivation in the tropics. All three tomato varieties showed adverse effects when exposed to drought but not when exposed to heat alone. The combination of drought and heat resulted in further injury. Where plants were well-watered, leaf temperatures were maintained at about 31 ⁰C regardless of ambient conditions. Elevated leaf temperatures were only observed when water supply and stomatal conductance were curtailed. On day 2, there were differences in the ability of the varieties to avoid overheating, with the best preforming variety having significantly higher stomatal conductance and significantly lower leaf
temperatures than the worst preforming variety. The varieties showed little variation in heat avoidance beyond day 2. After a week under stress, the largest differences among varieties were seen in chlorophyll content and in dark-adapted Fv/Fm measured in situ.
Heat avoidance under drought and well-watered conditions The regulation of leaf stomatal conductance is vital in preventing desiccation at the whole plant level, as well as for acquiring CO2 for photosynthesis. Stomata close in response to drought stress as a means for the plant to conserve water (Ashraf and Harris, 2013). At high temperatures, they remain open allowing evaporative cooling of the leaves (canopy temperature depression) (Salvucci and Crafts-Brandner, 2004; Sánchez et al., 2013; Feller, 2016). When leaf temperature starts to decrease, stomata may close or partially close to avoid excessive loss of water, especially if there is a water deficit. Prolonged exposure to a water deficit may cause heatinduced stomatal opening to shift towards a higher temperature (Feller and Vaseva, 2014). Our results show that for all varieties, plants in the HOT treatment had the highest stomatal conductance. Leaf surface temperature of this treatment was concomitantly lower than air temperature as evaporative cooling took effect (Fig. 1). Righi et al. (2012) also observed lowered leaf temperatures (relative to air temperature) in heat-stressed tomato plants grown without water restriction. In our study, evaporative cooling was so effective that the leaf temperature in wellwatered plants was decoupled from the air temperature. As expected, stomatal conductance was lowest and leaf temperature was highest in the DRY and the HOT+DRY treatments. All three varieties showed an ability to elevate stomatal conductance under higher air temperatures, but in all cases this was counteracted by stomatal closure when a water deficit was induced.
On day 9, plants from the HOT+DRY treatment had significantly higher leaf temperatures than the DRY treatment. In other words, the full effects of elevated mid-day air temperatures were only achieved when the heat treatment was combined with the drought treatment (i.e. HOT+DRY). This has important implications for designing heat screening protocols as, in many cases leaf temperature is not measured making a true assessment of heat tolerance difficult (Webber et al., 2015). Abdelmageed and Gruda (2009) evaluated the response of tomato varieties ranging in sensitivity to heat stress through examination of photosynthetic rates, but only took into account air temperature thereby discounting the ability of some genotypes to maintain higher stomatal conductance and lower leaf temperatures. Similarly, Zhang et al., (2014) studied the photosynthetic response of tomato to elevated air temperature without accounting for leaf temperature. Genotypic differences in our study were significant for stomatal conductance and leaf temperature on day 2. Hybrid 61 performed better than the other varieties and was able to maintain higher stomatal conductance and lower leaf temperatures even under drought conditions. The heat-tolerant Nagcarlang had lower stomatal conductance but higher leaf temperatures, which confirms its ability to tolerate heat, while Hybrid 61 seemed to be more effective at heat avoidance. Zhou et al. (2015) also observed that heat-tolerant lines of tomato seedlings under heat stress had significantly higher stomatal conductance than heat-sensitive lines and were better able to lower their leaf temperatures via evaporative cooling. By day 9 genotypic differences became obscured, indicating that these parameters should be measured sooner rather than later when testing for combined drought and heat avoidance.
Heat tolerance under drought and well-watered conditions
Prolonged exposure to drought can decrease photosynthesis by inducing stomatal closure, damaging the photosynthetic apparatus and producing changes in photosynthetic pigments (Farooq et al., 2009). Damage is most often detected by changes in Fv/Fm, which measures the maximum quantum efficiency of PSII (Maxwell and Johnson, 2000; Baker, 2008). Our results support this approach, as dark-adapted Fv/Fm measured in situ showed the largest differences between varieties. However, other fluorescence variables also provided useful information about the level of stress-induced damage, e.g. Fv/Fo (ratio of variable to minimum fluorescence) and Fq’/Fm’ (steady state quantum yield of PSII in the light). Fv/Fo, like Fv/Fm, was significantly reduced in the worst performing variety in the DRY treatment. Sharma et al. (2014) observed similar genotypic variations in the Fv/Fo and Fv/Fm of wheat (Triticum aestivum L.). However, they noted that Fv/Fo can be too sensitive in cases of mild stress and can enhance natural variations in control values, making Fv/Fm a better measure of abiotic stress. In our results, Fv/Fo values showed a similar pattern to Fv/Fm in all treatments although the variance was greater than for Fv/Fm. Fq’/Fm’ was significantly reduced in the DRY treatment for all of the varieties, but genotypic differences were not evident. This is an indication that the plants in this treatment were unable to efficiently make use of light for electron transport, likely due to stomatal limitations (imposed by drought stress) (Baker and Rosenqvist, 2004). Not many studies have examined the combined effects of water deficits and high temperatures on chlorophyll fluorescence variables. Most fluorescence variables examined in our study showed similar patterns in both of the drought treatments (i.e. DRY and HOT+DRY). Plants from both of the drought treatments had super-optimal leaf temperatures, which can lower photosynthetic rates due to metabolic inhibition of PSII and alterations of the thylakoid membranes (Willits and Peet, 2001; Morales et al., 2003; Aien et al., 2011). Substantial damage
to PSII is usually associated with Fv/Fm < 0.75; this was not evident in our study although a few individual plants were below this value, indicating the onset of severe stress. This indicates that the inhibition of PSII in plants under the DRY and the HOT+DRY treatments was temporary or reversible (Willits and Peet, 2001). Photosynthetic inhibition is known to be reversible when temperatures are slightly above optimal and irreversible when temperatures are severe (Salvucci and Crafts-Brandner, 2004; Ashraf and Harris, 2013). In our study, in situ leaf temperatures observed during mid-day hours were not maintained throughout the day and likely not held for long enough to induce irreversible damage. Ruling out severe damage to PSII, the significantly lower Fq’/Fm’ values observed in DRY and HOT+DRY can be attributed to down-regulation of downstream processes, that is, the rates of consumption of NADPH and ATP in CO2 assimilation (Baker and Rosenqvist, 2004; Chen et al., 2009). Stomatal conductance was clearly limited during these periods of mid-day heat stress and likely explains the reductions seen in Fq’/Fm’. Fq’/Fm’ may also have been impacted by other limitations to the carbon reactions, e.g. reduced activity of the thermosensitive enzyme rubisco activase has been found to limit the rate of the carbon reactions at high temperatures (Carmo-Silva et al., 2012; Carmo-Silva et al., 2015). As such, in all but the most sensitive variety, the impact of combined heat and drought on the quantum yield of PSII can be explained by limitation of the carbon reactions. As well as inhibition of the carbon reactions and of the photosystems, reduced photosynthesis under abiotic stress can occur due to reductions in chlorophyll content (Al Hassan et al., 2015). In our study, chlorophyll content showed significant responses to the treatments, with the combination of heat and drought producing additional reductions to that of drought alone (Fig. 2). Plant response to drought stress is variable as some species show a decrease in chlorophyll content (attributed mostly to accelerated degradation), while others show an
enhanced accumulation of chlorophyll (Jaleel et al., 2009; Ashraf and Harris, 2013; Zhou et al., 2015). We observed reductions of 4.8 %, 8.1 % and 6.8 % for Nagcarlang, Hybrid 61 and Moskvich, respectively, in the DRY treatment. These small reductions suggest that the large reductions seen in photosynthetic activities (inferred by significantly reduced Fq’/Fm’) in this treatment were due to other factors, particularly stomatal limitations. Al Hassan et al. (2015) also observed no significant reductions in the chlorophyll content of drought-stressed tomato (Cerasiforme) during mild drought. During heat stress, chlorophyll content may be a critical factor in determining the overall photosynthetic limitation. Both Nagcarlang and Moskvich had significantly reduced chlorophyll contents (these decreased by 18.5 % and 17.4 %, respectively). Only Hybrid 61 maintained high chlorophyll content across all treatments, with a comparatively small reduction of 4.9 % seen in the HOT+DRY treatment. Heat stress is known to either reduce chlorophyll biosynthesis or accelerate degradation (Ashraf and Harris, 2013). The significant reductions seen for Nagcarlang and Moskvich in the combined stress treatment in our study were likely because the leaf surface temperatures of plants in this treatment were high enough to impact chlorophyll turnover. In the absence of irreversibly injury, the ability to maintain a high chlorophyll content, as seen in Hybrid 61, allows the plant to restore leaf level photoassimilation once the immediate restriction on stomatal conductance is removed. This trait would offer considerable benefits in a variable climate where overheating and water limitation vary throughout the day. Zhou et al. (2015) observed increased chlorophyll content in heat-tolerant lines of tomato under super-optimal temperatures, but it was not clear why this occurred. Camejo et al. (2005) also observed increases and suggested that the increased chlorophyll content was an adaptation towards more
sun-type chloroplasts. These observations support the hypothesis that an ability to maintain an optimal chlorophyll content during moderate heat stress is a key heat tolerance trait in tomato. In vitro Fv/Fm values, following a heat-shock treatment, indicated that CONTROL plants were most affected by the heat shock treatment followed by plants from the HOT treatment, then the DRY treatment. Plants from the HOT+DRY treatment were significantly less affected by the in vitro heat shock. It is known that prior exposure to moderately high temperatures can help a plant to develop the ability to cope with subsequent, potentially lethal heat exposure (Farrell et al., 2006; Snyman and Cronjé, 2008; John-Bejai et al., 2013; Brestic and Zivcak, 2013). Overall, the response of in vitro Fv/Fm followed the order CONTROL < HOT < DRY < HOT+DRY; this is the same order as seen in mid-day leaf temperatures suggesting that acclimation had occurred. Plants from the combined stress treatment were the only ones where leaf surface temperatures were high on a regular basis and therefore they were the only ones that were truly acclimated to high temperatures. This acclimation meant that the in vitro heat shock did not severely reduce Fv/Fm in the tolerant varieties (Hybrid 61 and Nagcarlang). Moskvich, on the other hand, is known to be heat-sensitive and did not acclimate as well to in situ conditions, and thus showed a significantly greater reduction in in vitro Fv/Fm. This ability to acclimate to moderate heat stress is an essential trait if plants in dry soils are to survive sporadic periods of very high air temperatures.
Detecting genotypic variation in response to combined heat and drought A comparison between the varieties shows that the in vitro Fv/Fm results support the results for in situ Fv/Fm and Fv/Fo. As expected, Fq’/Fm’ was not a good indicator of genetic variation in heat or drought tolerance following prolonged stress. Only dark-adapted Fv/Fm showed
significances in the interaction between treatment and variety, highlighting its usefulness in identifying stress-resilient varieties. It can be particularly useful when selecting drought-tolerant lines as the effects of drought were clearer for this parameter. In vitro Fv/Fm and chlorophyll content also revealed significant differences between the best and the worst variety under HOT+DRY. Hybrid 61 had the highest Fv/Fm across all treatments throughout the acclimation period, as well as the lowest percent change in in vitro Fv/Fm and in chlorophyll content.This variety was also the best performer in terms of heat avoidance. Hybrid 61 is widely grown in tropical regions and is high yielding both in the open and under warm greenhouse conditions (Ali et al., 2015). Our results confirm that Hybrid 61 is well suited to growth under tropical conditions as it is heat-tolerant and, to a lesser extent, drought-tolerant.
Conclusions Under well-watered conditions, all three varieties were able to avoid overheating by increasing stomatal conductance, while under restricted water the heat-tolerant varieties were able to retain some canopy temperature depression over the first two days. Nonetheless, after 9 days of moderate drought, stomatal conductance was reduced to negligible levels in all varieties and the genotypic differences in leaf temperature were lost. At this point, the ability to acclimate and exhibit heat tolerance mechanisms became more important, as revealed by significantly higher values for in vitro Fv/Fm and chlorophyll content in the heat-tolerant varieties. In situ Fv/Fm was most affected by drought stress but in vitro Fv/Fm clearly highlights the impacts of heat stress and could be used to select heat-tolerant crop varieties.The ability to maintain chlorophyll content under moderate heat stress may be an under-explored mechanism for heat tolerance.
ACKNOWLEDGMENTS This work was supported by a Caribsave-INTASAVE Caribbean grant (CIRCA: Impacts and Resilience in Caribbean Agriculture, No. CDKN/RC28); and by The University of the West Indies (Grant No. CRP.4.MAR12.8). We thank Jane Deacon and Joshua Spiers for assistance with the work and Mike Jones for comments on the manuscript.
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S0176-1617(16)30126-2 http://dx.doi.org/doi:10.1016/j.jplph.2016.07.006 JPLPH 52405
To appear in: Received date: Revised date: Accepted date:
6-5-2016 6-7-2016 6-7-2016
Please cite this article as: Nankishore Alliea, Farrell Aidan D.The response of contrasting tomato genotypes to combined heat and drought stress.Journal of Plant Physiology http://dx.doi.org/10.1016/j.jplph.2016.07.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The response of contrasting tomato genotypes to combined heat and drought stress Alliea Nankishorea and Aidan D Farrellb* a
Department of Biology, University of Guyana, Guyana
b
Department of Life Sciences, University of the West Indies, St. Augustine Campus, Trinidad
and Tobago W.I.
Corresponding Author: * Aidan Farrell [email protected]
The response of contrasting tomato genotypes to combined heat and drought stress
ABSTRACT Efforts to maximize yields of food crops can be undermined by abiotic stress factors, particularly those related to climate change. Here, we use a range of physiological methods to detect the individual and combined effects of heat and drought stress on three contrasting varieties of tomato: Hybrid 61, Moskvich, and Nagcarlang. Seedlings were acclimated under the following treatment regimes: CONTROL (25-36 ⁰C; well-watered), DRY (25-36 ⁰C; 20 % field capacity), HOT (25-42 ⁰C; well-watered) and HOT+DRY (25-42 ⁰C; 20 % field capacity). In each treatment, stomatal conductance, leaf temperature, chlorophyll content, and several chlorophyll fluorescence variables (both in situ and in vitro following a heat shock treatment) were measured. Plants from the HOT treatment remained statistically similar to the CONTROL in all the measured parameters, while those from the DRY treatment and especially the HOT+DRY treatment showed clear effects of abiotic stress. Hybrid 61 showed considerable resilience to heat and drought stress compared to the other varieties, with significantly cooler leaves (one day after treatments imposed) and significantly higher Fv/Fm values both in situ and in vitro. The genotypic differences in resilience to heat stress were only apparent under water-limited conditions, highlighting the need to consider leaf temperature rather than air temperature when testing for tolerance to heat stress. The most effective parameters for discriminating genotypic variation in heat and drought stress were in vitro Fv/Fm and chlorophyll content.
Keywords: climate change; heat stress; water stress; plant breeding; leaf temperature; chlorophyll fluorescence.
INTRODUCTION
The global human population is currently growing at an unprecedented rate and is expected to remain on this trajectory for at least 35 years (Zargar et al., 2011; Meeks et al., 2013). An increasing population is associated with an increase in demand for food and this is projected to continue until food production has doubled (Howden et al., 2007; Bita and Gerats, 2013). However, efforts to grow and maximize yields of food crops can be undermined by climatic changes, such as increases in atmospheric temperature and decreases in precipitation (Zargar et al., 2011; Meeks et al., 2013; Eitzinger et al., 2015a, 2015b). Changes in these abiotic factors may induce physiological stress such as heat stress and drought stress in agronomically important plants. Drought stress is considered to be the most damaging abiotic stress to crop productivity (Foolad et al., 2003; Mir et al., 2012). High temperatures can also impact on crop productivity both directly and by exacerbating the effects of drought by promoting evapotranspiration (Farrell, 2014; Webber et al., 2015; Feller, 2016). These are major challenges in rainfed agriculture, especially in the arid and semi-arid regions of the tropics (Tomar and Kumar, 2004; Kulkarni and Deshpande, 2007). It is, therefore, vital to be able to identify and develop crop varieties that are resilient to abiotic stress so that crop productivity is not unduly affected (Foolad et al., 2003; Camejo et al., 2005; Bita et al., 2011; Feller and Vaseva, 2014). Tomato (Solanum lycopersicum L.) grows optimally at temperatures ranging from 20-30 ⁰C and is sensitive to extreme temperatures (Zhou et al., 2015) as well as water deficits (Petrozza et al., 2014). Tomato cultivation has increased in the tropics and subtropics where there is considerable risk from high temperature and drought periods. Previous studies have
demonstrated the drastic impacts of heat on tomato physiology (Sato et al., 2000; Singh et al., 2005; Camejo et al., 2006). Measures of stomatal conductance and leaf surface temperature are useful in determining the effects of stress on plant water relations and on their ability to avoid overheating, while measurements of chlorophyll content and chlorophyll fluorescence help to assess the level of stress-induced damage to photosynthetic structures and so indicate heat tolerance (Wahid et al., 2007; Farrell, 2014; Feller, 2016). Camejo et al. (2006) compared the response of heat-tolerant (Nagcarlang) and heat-sensitive (Amalia) varieties of tomato to heat stress after exposing seedlings to a heat shock at 45 ⁰C for 3 hours. Severe reductions in photosynthesis, stomatal conductance and chlorophyll content were observed in Amalia while Nagcarlang was unaffected. Recently, Zhou et al. (2015) observed similar reductions in these parameters in two heat-sensitive tomato varieties, relative to two heat-tolerant varieties. These same physiological parameters are seen to be impacted when tomato is grown under water deficit (Hayat et al., 2008; Zhang et al., 2011) although, in some cases, stomatal conductance and chlorophyll content have been seen to increase under heat stress while they typically decrease under drought stress (Zhou et al., 2015; Feller, 2016). Thus, there is a need to understand how the interaction between these physiological parameters contribute to genotypic variation in stress tolerance. Here, we use both in situ and in vitro physiological parameters measured in control and stress-acclimated tissue. In vitro stress testing provides a convenient technique to make uniform comparisons between varieties. In the case of high temperature stress, many studies have used an in vitro heat-shock method to estimate the overall thermal tolerance of leaf tissue (Willits and Peet, 2001; Camejo et al., 2005; Camejo et al., 2006). Exposing harvested leaf tissues to high temperatures in vitro for a relatively short period of time allows for identification of the lethal
temperature, above which there is disruption of metabolic processes and irreversible injury. Prior in situ acclimation to abiotic stress is an essential step, as it helps to enhance the ability of tissue to withstand future exposure to heat shock conditions (Mittler et al., 2011; Farrell, 2014). Mittler (2006) and Feller (2016) highlight the need to develop crops with enhanced resilience to a combination of different stresses and point to the potential negative and complex interaction between drought and high temperature. Drought-tolerant plants may not necessarily be tolerant to heat stress, and vice versa (Jagadish et al., 2011; Feller, 2016). Hence, we aim to examine the response to heat and/or drought in seedlings of three contrasting tomato varieties. Seedling response is evaluated using stomatal conductance, leaf surface temperature, chlorophyll content and chlorophyll fluorescence (measured in situ and in vitro). In particular, we seek to understand the role of heat avoidance, heat acclimation and heat tolerance processes in enabling resilient varieties to withstand the combined effects of heat and drought as experienced by plants under field conditions.
MATERIALS AND METHODS
Planting Materials The varieties used in our study were Nagcarlang, Hybrid 61 and Moskvich. Nagcarlang is heattolerant and is a wildtype, heirloom variety that originated in the Philippines (Camejo et al., 2005). Hybrid 61 is commonly grown in tropical regions and is known to be high-yielding and tolerant to harsh weather conditions, although it is little studied (Ali et al., 2015). In contrast,
Moskvich is sensitive to heat and is another heirloom variety with origins in Russia (Kamel et al., 2010). Tomato seeds were germinated at 22 ⁰C in the laboratory and then transplanted to 16ounce polystyrene cups with drainage holes, containing a 2:1 mixture of sharp sand and ProMix (Premier Tech Horticultural Inc., Quakertown, PA, USA). The plants were reared in a wellventilated, full-sun greenhouse at The University of the West Indies in St. Augustine, Trinidad, and were watered daily to field capacity. The daily photoperiod was approximately 12 hours and the typical mid-day sunlight within the greenhouse was 600 µmol/m2/s.
Acclimation At the 4-leaf stage, the varieties were arranged in a complete randomized block design, with 10 replicates per block and four treatments (CONTROL, HOT, DRY and HOT+DRY). Data loggers (HOBO Pro, Onset Corp., USA) were placed on each bench at canopy height to monitor air temperature and humidity. Each treatment was applied on adjacent benches as follows: 1. CONTROL: served as the control and were watered once a day with 150 mL of water. 2. HOT: bench was covered in a clear polythene sheet at a height of 1 m, which reduced the ventilation but allowed limited airflow from beneath the bench. Plants were watered once a day. 3. DRY: plants were partially deprived of water. Each pot received 20 mL of water per day, to maintain soil moisture at approximately 20 % of the soil field capacity. Soil volumetric water content was monitored 3 times per week from 6 replicates of each variety prior to rewatering, using a soil moisture meter (Fieldscout TDR 100 Soil Moisture Meter; Spectrum Technologies, Inc., Illinois).
4. HOT+DRY: HOT and DRY treatments were combined.
Over the course of the experiment on the uncovered benches (CONTROL and DRY), the mean daily air temperature was 29 ⁰C, with a mean daily range of 25-36 ⁰C, the mean daily relative humidity was 75 % with a mean daily range of 39-100 %, and the mean daily vapour pressure deficit was 0.9 kPa with a daily range of 0-2.9 kPa (HOBO Pro, Onset Corp., USA). On the covered benches (HOT and HOT + DRY) the mean daily air temperature was 30 ⁰C (range 25-42 ⁰C), the mean daily relative humidity was 73 % (range 30-100 %) and the mean daily vapour pressure deficit was 1.3 kPa (range 0-4.4 kPa). The different values on the covered benches were due to elevated mid-day temperatures, which were on average 5 ⁰C hotter than the uncovered benches. During and after acclimation, several physiological parameters were measured as set out in Table 1. All measurements were made mid-way along the leaf, using the first fully expanded leaf. Stomatal conductance was measured immediately after taking leaf temperature. The thermal camera was held at an angle of 45⁰ from the leaf, at a distance of approximately 0.45 m and at an emissivity of 0.98. Chlorophyll content was measured on a per area basis as the ratio of light absorbsion at 660 nm to that at 940 nm (SPAD equivalent ratio units; Zhu et al., 2012).
Table 1 Outline of the timing of all measurements Measurement
Instrument Used
Stomatal conductance
Leaf porometer (SC-1, Decagon Devices; Pullman; WA, USA)
Days from Treatment Time of Measurement 1 2 3 4 5 6 7 8 9 10 11 12 14 Twice weekly during mid-day hours (10:0014:00 h)
Leaf surface temperature
Thermal camera (TI400, Fluke, USA)
Twice weekly during mid-day hours
Chlorophyll content
Chlorophyll meter (atLEAF+, FT Green LLC, Detroit, USA)
Once weekly after midday hours
Dark adapted chlorophyll fluorescence
Pulse modulated chlorophyll fluorometer (Mini-PAM; Walz; Effeltrich, Germany)
Once weekly at least 1 hour after dark (20:0021:30 h)
Light adapted chlorophyll fluorescence
Pulse modulated chlorophyll fluorometer (Mini-PAM; Walz; Effeltrich, Germany)
Once weekly during mid-day hours
In vitro chlorophyll fluorescence
Pulse modulated chlorophyll fluorometer (Mini-PAM; Walz; Effeltrich, Germany)
Once post-acclimation
A modulated chlorophyll fluorometer was used to estimate the steady state quantum yield of photosystem II in the light (at mid-day; Fq’/Fm’) and the maximum quantum yield of photosystem II following dark-adaptation (one hour after nightfall; Fv/Fm) (Baker, 2008).
Laboratory Measurements After acclimation, the plants were transported from the greenhouse to the laboratory and placed in a dark room for 40 minutes in order to dark adapt. One leaf was harvested from each plant and rinsed in 20 mL distilled water. In vitro Fv/Fm was obtained for each leaf using a pulse modulated chlorophyll fluorometer (Mini-PAM; Walz; Effeltrich, Germany). Each leaf was then placed in a labeled heat-shock vial containing 20 mL de-ionized water and left in a preheated (43 ⁰C) water bath for 1 hour, after which fluorescence measurements were repeated. The difference between measurements taken before and after the heat-shock were expressed as a percentage: (fluorescence after heat treatment / fluorescence before heat treatment × 100). A pilot study with
control plants showed that leaves exposed to a room temperature (26 ⁰C) treatment were unaffected by the in vitro incubation.
Statistical Analysis Data were analyzed using the statistical software R (Version 3.2.1, R Foundation for Statistical Computing, Vienna, Austria) for two-way analysis of variance (ANOVA) and post-hoc Fisher’s Least Significant Difference (LSD). Plants were grouped with treatment and variety as factors. Differences were considered as significant when p < 0.05.
RESULTS
Stomatal conductance Stomatal conductance after 1 day of exposure to heat and/or drought stress (Fig. 1A) differed significantly among treatments (p < 0.001) and among varieties (p < 0.05), but the interaction between these two factors was not significant (p > 0.05). Plants under the HOT treatment had elevated stomatal conductance, although the differences were not significant for individual varieties. Conductance readings for plants from the DRY treatment were lower than those from the CONTROL treatment, while those from the HOT+DRY treatment were significantly lower than the HOT treatment. Of the three varieties, Hybrid 61 showed the highest conductance across all treatments.
Fig. 1. Stomatal conductance (SC; mmol m-2s-1) and leaf temperature (LT; ⁰C) after 2 days (A and C, respectively) and 9 days (B and D, respectively) of stress. Bars represent means + standard errors, n = 10. Bars that do not have a common letter are significantly different within each sub-figure according to Fisher’s LSD test.
On day 9, plants from the CONTROL and the HOT treatments showed higher conductance levels than seen on the previous measurement day while those from the DRY and the HOT+DRY treatments were lower (Fig. 1B). There were significant differences between treatments (p < 0.001) but not between the other factors. Conductance readings for plants from the DRY treatment were significantly lower than those from the CONTROL treatment, while those from the HOT+DRY treatment were significantly lower than the HOT treatment. Differences between treatments were more pronounced than on day 2, but genotypic differences were less apparent.
Leaf surface temperature After 1 day of exposure to heat and/or drought stress, plants showed significant differences among treatments (p < 0.001) and near significant differences among varieties (p = 0.058). The interaction between these two factors was not significant. Plants from the CONTROL and the HOT treatments had similar leaf temperatures (Fig. 1C) but these were significantly lower than those from the DRY and the HOT+DRY treatments, which were also similar to each other. In the HOT+DRY, Hybrid 61, which had the highest stomatal conductance, also had the lowest leaf temperature (significantly lower than Nagcarlang). On day 9, differences in leaf surface temperature among treatments remained significant (p < 0.001), while differences among varieties and for the interaction were not significant. Plants from the CONTROL and the HOT treatments still maintained similar leaf temperatures. Plants in the DRY treatment, with negligible stomatal conductance, remained significantly hotter than those in the CONTROL treatment, while plants in the HOT+DRY treatment were significantly hotter than all other treatments (Fig. 1D).
Chlorophyll content After 10 days of exposure to the stress conditions, the chlorophyll content of leaves showed significant differences among treatments (p < 0.001) and varieties (p < 0.05), but no significant interaction. For all three varieties, plants in the DRY treatment had less chlorophyll than those in the CONTROL treatment (not significant). In the HOT+DRY treatment, Nagcarlang and Moskvich had lower values than in the other treatments, while Hybrid 61 maintained its chlorophyll content in all treatments (Fig. 2).
Fig. 2. Chlorophyll content (ratio units) after 10 days of stress. Bars represent means + standard errors, n = 10. Bars that do not have a common letter are significantly different according to Fisher’s LSD test.
In situ chlorophyll fluorescence After 10 days of exposure to heat and/or drought stress, Fo was significantly different among varieties (p < 0.001) but not among treatments or in the interaction (Fig. 3A). Fm was significantly different among treatments (p < 0.001), with lower values seen under DRY and HOT+DRY conditions, but not among varieties or in the interaction (Fig. 3B). Fv/Fo was significantly different among treatments (p < 0.05) and varieties (p < 0.001), but not in the interaction, while Fv/Fm was significant for all factors. For Nagcarlang and Moskvich, plants in
the DRY treatment had lower Fv/Fo and Fv/Fm values than in the CONTROL treatment (significantly lower for Moskvich), while Hybrid 61 maintained high values across all treatments. The HOT+DRY treatment also showed lower Fv/Fo and Fv/Fm values for Moskvich compared to CONTROL and HOT plants, although these were not lower than the values from the DRY treatment (Figs. 3C and 3D). Fq’/Fm’ was significantly different among treatments (p < 0.001), but not among varieties or in the interaction. Plants from the DRY and HOT+DRY treatments were significantly lower than the CONTROL but similar to each other (Fig. 3F). No genotypic differences were apparent.
Fig. 3. Sub-figures A, B, C, D and F represent dark-adapted mean Fo, Fm, Fv/Fo, Fv/Fm and light adapted mean Fq’/Fm’, respectively, after 10 days of stress, while sub-figure E illustrates mean difference in Fv/Fm after in vitro heat-shock. Bars represent means + standard errors, n =
10. Bars that do not have a common letter are significantly different within each sub-figure according to Fisher’s LSD test.
In vitro chlorophyll fluorescence Following exposure to 43 ⁰C for 1 hour, in vitro fluorescence varied significantly among treatments (p < 0.001), but not among varieties or in the interaction between these two factors. Plants from the HOT and the DRY treatments tended to withstand the heat-shock better than plants from the CONTROL (only significant for Nagcarlang). Plants from the HOT+DRY treatment maintained significantly higher post heat-shock Fv/Fm values when compared to all other treatments (Fig. 3E). Hot acclimated Hybrid 61 plants were significantly more tolerant to heat shock than Moskvich.
DISCUSSION
Our treatments included relatively brief periods of very high temperatures occurring at mid-day, similar to conditions experienced under cultivation in the tropics. All three tomato varieties showed adverse effects when exposed to drought but not when exposed to heat alone. The combination of drought and heat resulted in further injury. Where plants were well-watered, leaf temperatures were maintained at about 31 ⁰C regardless of ambient conditions. Elevated leaf temperatures were only observed when water supply and stomatal conductance were curtailed. On day 2, there were differences in the ability of the varieties to avoid overheating, with the best preforming variety having significantly higher stomatal conductance and significantly lower leaf
temperatures than the worst preforming variety. The varieties showed little variation in heat avoidance beyond day 2. After a week under stress, the largest differences among varieties were seen in chlorophyll content and in dark-adapted Fv/Fm measured in situ.
Heat avoidance under drought and well-watered conditions The regulation of leaf stomatal conductance is vital in preventing desiccation at the whole plant level, as well as for acquiring CO2 for photosynthesis. Stomata close in response to drought stress as a means for the plant to conserve water (Ashraf and Harris, 2013). At high temperatures, they remain open allowing evaporative cooling of the leaves (canopy temperature depression) (Salvucci and Crafts-Brandner, 2004; Sánchez et al., 2013; Feller, 2016). When leaf temperature starts to decrease, stomata may close or partially close to avoid excessive loss of water, especially if there is a water deficit. Prolonged exposure to a water deficit may cause heatinduced stomatal opening to shift towards a higher temperature (Feller and Vaseva, 2014). Our results show that for all varieties, plants in the HOT treatment had the highest stomatal conductance. Leaf surface temperature of this treatment was concomitantly lower than air temperature as evaporative cooling took effect (Fig. 1). Righi et al. (2012) also observed lowered leaf temperatures (relative to air temperature) in heat-stressed tomato plants grown without water restriction. In our study, evaporative cooling was so effective that the leaf temperature in wellwatered plants was decoupled from the air temperature. As expected, stomatal conductance was lowest and leaf temperature was highest in the DRY and the HOT+DRY treatments. All three varieties showed an ability to elevate stomatal conductance under higher air temperatures, but in all cases this was counteracted by stomatal closure when a water deficit was induced.
On day 9, plants from the HOT+DRY treatment had significantly higher leaf temperatures than the DRY treatment. In other words, the full effects of elevated mid-day air temperatures were only achieved when the heat treatment was combined with the drought treatment (i.e. HOT+DRY). This has important implications for designing heat screening protocols as, in many cases leaf temperature is not measured making a true assessment of heat tolerance difficult (Webber et al., 2015). Abdelmageed and Gruda (2009) evaluated the response of tomato varieties ranging in sensitivity to heat stress through examination of photosynthetic rates, but only took into account air temperature thereby discounting the ability of some genotypes to maintain higher stomatal conductance and lower leaf temperatures. Similarly, Zhang et al., (2014) studied the photosynthetic response of tomato to elevated air temperature without accounting for leaf temperature. Genotypic differences in our study were significant for stomatal conductance and leaf temperature on day 2. Hybrid 61 performed better than the other varieties and was able to maintain higher stomatal conductance and lower leaf temperatures even under drought conditions. The heat-tolerant Nagcarlang had lower stomatal conductance but higher leaf temperatures, which confirms its ability to tolerate heat, while Hybrid 61 seemed to be more effective at heat avoidance. Zhou et al. (2015) also observed that heat-tolerant lines of tomato seedlings under heat stress had significantly higher stomatal conductance than heat-sensitive lines and were better able to lower their leaf temperatures via evaporative cooling. By day 9 genotypic differences became obscured, indicating that these parameters should be measured sooner rather than later when testing for combined drought and heat avoidance.
Heat tolerance under drought and well-watered conditions
Prolonged exposure to drought can decrease photosynthesis by inducing stomatal closure, damaging the photosynthetic apparatus and producing changes in photosynthetic pigments (Farooq et al., 2009). Damage is most often detected by changes in Fv/Fm, which measures the maximum quantum efficiency of PSII (Maxwell and Johnson, 2000; Baker, 2008). Our results support this approach, as dark-adapted Fv/Fm measured in situ showed the largest differences between varieties. However, other fluorescence variables also provided useful information about the level of stress-induced damage, e.g. Fv/Fo (ratio of variable to minimum fluorescence) and Fq’/Fm’ (steady state quantum yield of PSII in the light). Fv/Fo, like Fv/Fm, was significantly reduced in the worst performing variety in the DRY treatment. Sharma et al. (2014) observed similar genotypic variations in the Fv/Fo and Fv/Fm of wheat (Triticum aestivum L.). However, they noted that Fv/Fo can be too sensitive in cases of mild stress and can enhance natural variations in control values, making Fv/Fm a better measure of abiotic stress. In our results, Fv/Fo values showed a similar pattern to Fv/Fm in all treatments although the variance was greater than for Fv/Fm. Fq’/Fm’ was significantly reduced in the DRY treatment for all of the varieties, but genotypic differences were not evident. This is an indication that the plants in this treatment were unable to efficiently make use of light for electron transport, likely due to stomatal limitations (imposed by drought stress) (Baker and Rosenqvist, 2004). Not many studies have examined the combined effects of water deficits and high temperatures on chlorophyll fluorescence variables. Most fluorescence variables examined in our study showed similar patterns in both of the drought treatments (i.e. DRY and HOT+DRY). Plants from both of the drought treatments had super-optimal leaf temperatures, which can lower photosynthetic rates due to metabolic inhibition of PSII and alterations of the thylakoid membranes (Willits and Peet, 2001; Morales et al., 2003; Aien et al., 2011). Substantial damage
to PSII is usually associated with Fv/Fm < 0.75; this was not evident in our study although a few individual plants were below this value, indicating the onset of severe stress. This indicates that the inhibition of PSII in plants under the DRY and the HOT+DRY treatments was temporary or reversible (Willits and Peet, 2001). Photosynthetic inhibition is known to be reversible when temperatures are slightly above optimal and irreversible when temperatures are severe (Salvucci and Crafts-Brandner, 2004; Ashraf and Harris, 2013). In our study, in situ leaf temperatures observed during mid-day hours were not maintained throughout the day and likely not held for long enough to induce irreversible damage. Ruling out severe damage to PSII, the significantly lower Fq’/Fm’ values observed in DRY and HOT+DRY can be attributed to down-regulation of downstream processes, that is, the rates of consumption of NADPH and ATP in CO2 assimilation (Baker and Rosenqvist, 2004; Chen et al., 2009). Stomatal conductance was clearly limited during these periods of mid-day heat stress and likely explains the reductions seen in Fq’/Fm’. Fq’/Fm’ may also have been impacted by other limitations to the carbon reactions, e.g. reduced activity of the thermosensitive enzyme rubisco activase has been found to limit the rate of the carbon reactions at high temperatures (Carmo-Silva et al., 2012; Carmo-Silva et al., 2015). As such, in all but the most sensitive variety, the impact of combined heat and drought on the quantum yield of PSII can be explained by limitation of the carbon reactions. As well as inhibition of the carbon reactions and of the photosystems, reduced photosynthesis under abiotic stress can occur due to reductions in chlorophyll content (Al Hassan et al., 2015). In our study, chlorophyll content showed significant responses to the treatments, with the combination of heat and drought producing additional reductions to that of drought alone (Fig. 2). Plant response to drought stress is variable as some species show a decrease in chlorophyll content (attributed mostly to accelerated degradation), while others show an
enhanced accumulation of chlorophyll (Jaleel et al., 2009; Ashraf and Harris, 2013; Zhou et al., 2015). We observed reductions of 4.8 %, 8.1 % and 6.8 % for Nagcarlang, Hybrid 61 and Moskvich, respectively, in the DRY treatment. These small reductions suggest that the large reductions seen in photosynthetic activities (inferred by significantly reduced Fq’/Fm’) in this treatment were due to other factors, particularly stomatal limitations. Al Hassan et al. (2015) also observed no significant reductions in the chlorophyll content of drought-stressed tomato (Cerasiforme) during mild drought. During heat stress, chlorophyll content may be a critical factor in determining the overall photosynthetic limitation. Both Nagcarlang and Moskvich had significantly reduced chlorophyll contents (these decreased by 18.5 % and 17.4 %, respectively). Only Hybrid 61 maintained high chlorophyll content across all treatments, with a comparatively small reduction of 4.9 % seen in the HOT+DRY treatment. Heat stress is known to either reduce chlorophyll biosynthesis or accelerate degradation (Ashraf and Harris, 2013). The significant reductions seen for Nagcarlang and Moskvich in the combined stress treatment in our study were likely because the leaf surface temperatures of plants in this treatment were high enough to impact chlorophyll turnover. In the absence of irreversibly injury, the ability to maintain a high chlorophyll content, as seen in Hybrid 61, allows the plant to restore leaf level photoassimilation once the immediate restriction on stomatal conductance is removed. This trait would offer considerable benefits in a variable climate where overheating and water limitation vary throughout the day. Zhou et al. (2015) observed increased chlorophyll content in heat-tolerant lines of tomato under super-optimal temperatures, but it was not clear why this occurred. Camejo et al. (2005) also observed increases and suggested that the increased chlorophyll content was an adaptation towards more
sun-type chloroplasts. These observations support the hypothesis that an ability to maintain an optimal chlorophyll content during moderate heat stress is a key heat tolerance trait in tomato. In vitro Fv/Fm values, following a heat-shock treatment, indicated that CONTROL plants were most affected by the heat shock treatment followed by plants from the HOT treatment, then the DRY treatment. Plants from the HOT+DRY treatment were significantly less affected by the in vitro heat shock. It is known that prior exposure to moderately high temperatures can help a plant to develop the ability to cope with subsequent, potentially lethal heat exposure (Farrell et al., 2006; Snyman and Cronjé, 2008; John-Bejai et al., 2013; Brestic and Zivcak, 2013). Overall, the response of in vitro Fv/Fm followed the order CONTROL < HOT < DRY < HOT+DRY; this is the same order as seen in mid-day leaf temperatures suggesting that acclimation had occurred. Plants from the combined stress treatment were the only ones where leaf surface temperatures were high on a regular basis and therefore they were the only ones that were truly acclimated to high temperatures. This acclimation meant that the in vitro heat shock did not severely reduce Fv/Fm in the tolerant varieties (Hybrid 61 and Nagcarlang). Moskvich, on the other hand, is known to be heat-sensitive and did not acclimate as well to in situ conditions, and thus showed a significantly greater reduction in in vitro Fv/Fm. This ability to acclimate to moderate heat stress is an essential trait if plants in dry soils are to survive sporadic periods of very high air temperatures.
Detecting genotypic variation in response to combined heat and drought A comparison between the varieties shows that the in vitro Fv/Fm results support the results for in situ Fv/Fm and Fv/Fo. As expected, Fq’/Fm’ was not a good indicator of genetic variation in heat or drought tolerance following prolonged stress. Only dark-adapted Fv/Fm showed
significances in the interaction between treatment and variety, highlighting its usefulness in identifying stress-resilient varieties. It can be particularly useful when selecting drought-tolerant lines as the effects of drought were clearer for this parameter. In vitro Fv/Fm and chlorophyll content also revealed significant differences between the best and the worst variety under HOT+DRY. Hybrid 61 had the highest Fv/Fm across all treatments throughout the acclimation period, as well as the lowest percent change in in vitro Fv/Fm and in chlorophyll content.This variety was also the best performer in terms of heat avoidance. Hybrid 61 is widely grown in tropical regions and is high yielding both in the open and under warm greenhouse conditions (Ali et al., 2015). Our results confirm that Hybrid 61 is well suited to growth under tropical conditions as it is heat-tolerant and, to a lesser extent, drought-tolerant.
Conclusions Under well-watered conditions, all three varieties were able to avoid overheating by increasing stomatal conductance, while under restricted water the heat-tolerant varieties were able to retain some canopy temperature depression over the first two days. Nonetheless, after 9 days of moderate drought, stomatal conductance was reduced to negligible levels in all varieties and the genotypic differences in leaf temperature were lost. At this point, the ability to acclimate and exhibit heat tolerance mechanisms became more important, as revealed by significantly higher values for in vitro Fv/Fm and chlorophyll content in the heat-tolerant varieties. In situ Fv/Fm was most affected by drought stress but in vitro Fv/Fm clearly highlights the impacts of heat stress and could be used to select heat-tolerant crop varieties.The ability to maintain chlorophyll content under moderate heat stress may be an under-explored mechanism for heat tolerance.
ACKNOWLEDGMENTS This work was supported by a Caribsave-INTASAVE Caribbean grant (CIRCA: Impacts and Resilience in Caribbean Agriculture, No. CDKN/RC28); and by The University of the West Indies (Grant No. CRP.4.MAR12.8). We thank Jane Deacon and Joshua Spiers for assistance with the work and Mike Jones for comments on the manuscript.
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