I Zandalinas Sara (Orcid ID: 0000-0002-1256-9371)

Mittler Ron (Orcid ID: 0000-0003-3192-7450)

Meta-analysis of drought and heat stress combination impact on crop yield and yield components

Itay Cohen, Sara I. Zandalinas, Clayton Huck, Felix B. Fritschi and Ron Mittler*

Division of Plant Sciences and Department of Biochemistry, College of Agriculture Food and Natural
Resources, and Interdisciplinary Plant Group. Christopher S. Bond Life Sciences Center University of
Missouri. 1201 Rollins St, Columbia, MO 65201

Correspondence

*Corresponding author: mittlerr@[Link]

Abstract

Episodes of prolonged drought coupled with heat waves (i.e., drought and heat combination) can have a
devastating impact on agricultural production and crop yield. It is therefore not surprising that improving
tolerance to drought and heat combination has been a major goal for breeders and biotech companies.
Although much is known about the physiological and molecular responses of vegetative tissues to a
combination of drought and heat stress, less is known about the impact of this stress combination on yield
and different yield components. Here, we used a meta-analysis approach to synthesize results from over
120 published case studies of crop responses to combined drought and heat stress. Our findings reveal that
drought and heat stress combination significantly impacts yield by decreasing harvest index, shortening the
life cycle of crops, and altering seed number, size and composition. Furthermore, these impacts are more
severe when the stress combination is applied during the reproductive stage of plants. We further identify
differences in how legumes and cereals respond to the stress combination and reveal that utilizing C3 or C4
metabolism may not provide an advantage to plants during stress combinations. Taken together our study

This is the author manuscript accepted for publication and has undergone full peer review but
has not been through the copyediting, typesetting, pagination and proofreading process, which
may lead to differences between this version and the Version of Record. Please cite this article
as doi: 10.1111/ppl.13203

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highlights a need to focus future studies, as well as breeding efforts, on crop responses to drought and heat
combination at the reproductive stage of different crop species.

Abbreviations: DS, drought stress; HI, harvest index; HS, heat stress; SNPs, single nucleotide
polymorphisms; HS + DS, combination of heat and drought stresses.

Introduction

Cereals and legumes are grown on more than 160 million hectares of non-irrigated (rain-fed) land
worldwide, supporting millions of lives in many different countries (Dwivedi et al. 2018). While growing
on non-irrigated lands, these crops are potentially subjected to different environmental stress conditions
such as drought, heat stress and their combination. Heat waves combined with acute and prolonged drought
stress can have a devastating outcome for agriculture, as well as economic and social stability, primarily
impacting drylands used for grain production worldwide (Ciais et al. 2005, Mittler 2006, Chen et al. 2019,
Zandalinas et al. 2020). The 2003 drought and heat episode across Europe, for example, caused a 30%
reduction in agricultural production (Ciais et al. 2005). Combined instances of drought and heat wave have
considerably increased in the United States during the period of 1990-2010 compared to 1960-1980
(Mazdiyasni and AghaKouchak 2015), with climate models predicting that these episodes will further
intensify (Lobell and Gourdji 2012, Lawas et al. 2018b); with an average increase of 2°C already recorded
for production regions of major crops such as wheat (Triticum aestivum), rice (Oryza sativa) and maize
(Zea mays). Models further predict that more significant yield decreases in the 2nd half of this century will
take place in tropical areas compared to temperate regions (Challinor et al. 2014). Field studies with soybean
(Glycine max) demonstrated that episodes of drought and heat negate the boost C3 plants receive from
growing in a CO2 enriched environment (Gray et al. 2016). This recent insight from field experimentation
imposing combined heat and drought demonstrates the likelihood of negative impacts from interacting
global change factors on other key global commodity crops such as wheat and maize in their primary
production regions.

Combined heat and drought stresses were found to negatively impact the yield of major crop plants
including legumes such as soybean, chickpea (Cicer arietinum) and lentil (Lens culinaris) (Dornbos Jr. and
Mullen 1991, Awasthi et al. 2014, Sehgal et al. 2017), as well as cereals such as wheat, maize and rice

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(Prasad et al. 2011, Cairns et al. 2013, Obata et al. 2015, Lawas et al. 2018a), which are the mainstay of
global food and feed supply in terms of both calorie and protein intake. A recent study in India showed that
supplying field crops such as wheat with irrigation can offset some of the yield reductions associated with
water-limited, heat stressed plants (Zaveri and Lobell 2019). Nonetheless, the use of fresh water to irrigate
wheat is unsustainable and aggravates conditions of water scarcity similar to the ones faced by India during
the summer of 2019 (Shah and Narain 2019).

Most studies in both model and crop plants have focused on the effects of drought or heat on yield as single
stress factors, or focused on the effects of combined stresses during the vegetative stages without
emphasizing how grain yields are impacted (Prasad et al. 2011, Obata et al. 2015, Lawas et al. 2018b). This
is despite the prevalence of combined heat and drought periods in field grown plants during reproductive
growth, and the fact that we can’t extrapolate plant responses to stress combinations simply by the addition
of responses to the two single stresses that comprise the stress combination (Rizhsky et al. 2004, Mittler
2006, Suzuki et al. 2014). In vegetative tissues, combined heat and drought negatively impacts vital
physiological plant processes such as stomatal conductance, photosynthesis, and respiration (Rizhsky et al.
2002, 2004, Zandalina et al. 2020). Furthermore, carbon balance is disturbed under drought and heat
combination due to increased carbohydrate demand for cellular respiration taking place simultaneously
with the decrease in photosynthesis (Foyer et al. 2009, Yu et al. 2012). Indeed, heat stress can cause a
negative whole plant carbon balance even under relatively mild water deficit conditions (Zhao et al. 2013).

Drought and heat combination during the reproductive stages are more detrimental by far to crop yields
because flower, ovary or seed abortion are irreversible processes, while the photosynthesis machinery can
recover once the stress is removed (Mahalingam and Bregitzer 2019, Balfagón et al. 2019). Drought and
heat combination can also shorten the life cycle, overall carbon assimilation and grain filling period in crops
(Awasthi et al. 2014). Additionally, this stress combination negatively impacts a variety of yield
components such as harvest index (HI), seed number and single seed size (Rollins et al. 2013, Sehgal et al.
2017).

Due to the complexities of properly controlling four different environments (control, heat, drought, and
drought and heat), large scale phenotyping of crop plants subjected to drought and heat combination are
rare, and until now reported only for maize (Cairns et al. 2013; stress was applied during anthesis),

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groundnut (Arachis hypogea; Hamidou et al. 2013; Stress was applied at the onset of flowering) and wheat
(Li et al. 2019; stress was applied one week after anthesis). Drought and heat combination studies performed
on large populations of crop plants (maize and wheat) suggest that genotypes with tolerance to heat or
drought as a single stress do not necessarily exhibit tolerance to the combination of these stresses (Cairns
et al. 2013, Qaseem et al. 2019). These findings are consistent with previous evidence that transcripts
upregulated in response to stress combination do not necessarily increase in response to the two single stress
conditions that comprise the stress combination (Mittler 2006). Furthermore, single nucleotide
polymorphisms (SNPs) found to be significantly associated with grain yield during a single stress do not
overlap with the ones observed under combined stress conditions (Yuan et al. 2019). These results further
emphasize the original findings of Rizhsky et al. (2002, 2004), that we cannot extrapolate the plants’
responses to stress combination from its responses to each of the two different stresses that comprise the
stress combination. Though previous studies suggested that a transgenic approach can mitigate the effects
of drought or heat stress applied individually (e.g., Ambavaram et al. 2014), published work that
demonstrates mitigation of the impact of combined heat and drought stress, to the best of our knowledge,
is limited to a single study in cotton (Gossypuim hirsutum) by Mishra et al. (2017).

Although previous reviews summarized the effects of drought and heat combination on different model and
crop plants (Mittler 2006, Prasad et al. 2008, Barnabás et al. 2008, Suzuki et al. 2014, Lawas et al. 2018b,
Sehgal et al. 2018), a comprehensive meta-analysis of crop responses to combined drought and heat stresses
has not been conducted. Here we synthesized results from 126 different case studies (Table S1) obtained
from 49 different publications (Table S2) reporting the impacts of drought, heat, and their combination on
yield, yield components, and seed composition.

Materials and methods

In the winter of 2019, a variety of key word searches were performed using different scientific bibliographic
tools such as Google scholar, Web of Science, Pubmed and AGRICOLA focused on the effects of drought
and heat combination on plants. In addition, lists of cited references in relevant reviews were surveyed
(Mittler 2006, Prasad et al. 2008, Barnabás et al. 2008, Suzuki et al. 2014, Lawas et al. 2018b, Sehgal et al.

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2018). Overall, we extracted and analyzed 126 different case studies taken from 49 different peer-reviewed
publications (Tables S1, S2).

Several factors were taken into consideration when analyzing the published data. These included: Plant
species, type of photosynthetic pathway used (C3 or C4; no reports on CAM or C3-C4 or C3-CAM
intermediates were found), rooting environment (pots or field environment), plant family (most plants were
either in the cereals or legumes families; however, few studies included crops in the Brassicaceae or
Solanum families), and the developmental stage of studied plants at the time stress was applied (vegetative
or reproductive). In addition, experiments that used a short (less than 12 hour) heat treatment or used fewer
than 4 replications per treatment were omitted.

Meta-analysis was only performed on data acquired from published, peer-reviewed studies that met several
specific criteria. Many publications displayed results from physiological measurements such as gas
exchange and water potential; however, we primarily focused on studies that presented data on yield and
yield components. To be included in the analyses, studies had to report on all four conditions: control, heat
stress, drought and drought and heat stress combination. Short term or small-scale experiments were
excluded. Moreover, results from the model plant Arabidopsis were also excluded. When data was
presented in a bar graph, the image was digitized and WebPlotDigitizer 4.2 software was used to extract
values. In some cases, the harvest index (HI) parameter was not explicitly reported, therefore we calculated
it from the data on yield and overall biomass (Erice et al. 2014). Parameters subjected to the meta-analysis
included: yield, HI, single seed size, seed protein content, seed starch content, and time needed to complete
life cycle. Data is presented as the mean of all relevant cases. Since comparing yield and yield parameters
from field and greenhouse experiments is not entirely valid, and as crop species varied greatly in their yield,
we normalized results from all studies to percentage (%) of the results of the control group for all
parameters. Since some publications reported on more than one genotype, we included these genotypes as
separate case studies. To determine statistical significance, combined case studies were subjected to a one-
or two-way ANOVA followed by Tukey’s test.

Results

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Impact of drought and heat stress combination on yield and yield components

Comparing the impact of drought and heat stress combination to that of drought or heat stresses applied
individually for all cases studied, revealed that the combination of drought and heat stress had a more severe
impact on yield compared to drought or heat alone (Figs. 1 and S1). Compared to controls, crops subjected
to heat or drought stresses applied as single stress factor displayed a 33 and 48% yield reduction,
respectively, whereas the average yield reduction in response to a combination of drought and heat stress
was 65% (Fig. 1A). This impact of stress combination on yield could manifest itself in a number of different
ways. We therefore compared the impact of stress combination on different yield components to that of
each of the stresses applied individually.

Harvest index is a key indicator of biomass partitioning between vegetative and reproductive organs in crop
plants. It is defined as the total weight of grain divided by the total weight of above ground biomass. As
highlighted in Figs. 1B and S2, when different crops were exposed to heat or drought, their HI decreased
by 28 and 27%, respectively. Interestingly, the combination of drought and heat further impacted HI and
reduced it by 53%. This result suggests that the drought and heat stress combination may shorten the life
cycle of annual crops and drive them to produce seeds earlier, and most likely in reduced numbers and/or
size, with less vegetative tissues produced in the process. Indeed, as shown in Fig. 1C (calculated for all
studies that measured the length of life cycle of crops), the combination of drought and heat stress shortened
the life cycle of most crops by an average of more than 40%. Interestingly, the impact of drought on life
cycle (a decrease of about 20%) was significantly different than that of heat (a decrease of about 30%)
revealing that drought had the least impact on shortening the life cycle of crops compared to heat or drought
and heat combination (with drought and heat combination having the maximal effect).

The impact of drought and heat stress combination on seed number, as well as seed starch and protein
content

Plants that display a shortened life cycle often also exhibit a reduced number of seeds per reproductive unit
(e.g., pod, spike, panicle, ear). Heat and drought as single stresses reduced seed numbers in each
reproductive unit, and their combination further magnified the impact, resulting in a 32% decrease in seed
number per reproductive unit (Fig. 2A).

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Seed composition is determined by many factors, including the environmental conditions during the seed
filling processes, as well as the overall biomass of the plant, accumulated during the vegetative stage. Thus,
we reviewed the impacts of drought, heat and drought and heat combinations on seed starch and protein
contents. As shown in Fig. 2B, the reduction in starch content by the stress combination (60%) was about
double that observed in response to heat or drought individually (30%). In contrast, seed protein content
was elevated compared to control in all three stress treatments (Fig. 2C). However, while drought or heat
caused a moderate increase in protein content of about 10-15%, the combination of drought and heat
increased seed protein content by more than 30%. Although the reason for the increase in seed protein
content during stress combination is unknown at present, it is possible that this increase reflects a decrease
in overall seed water content and/or a compensatory mechanism to the decrease in starch content. Overall,
the findings presented in Fig. 2B,C demonstrate that, in addition to the more pronounced decrease in yield
and number of seeds per reproductive unit (Figs. 1A and 2A), the drought and heat stress combination also
amplified the impact on seed composition compared to the effect of heat or drought individually (Fig.
2B,C).

The negative impact of stress combination is greater during the reproductive stage

The impact of stress combination on yield, HI, life cycle and seed composition (Figs. 1 and 2) could result
from the stress combination impact on overall biomass through suppression of photosynthesis, from its
effects on plant reproductive processes such as fertilization and/or abortion, and/or from its effects on seed
filling. To explore the effects of stress combination relative to developmental stage, we divided the data set
into experiments that subjected crops to stress during the vegetative or the reproductive stages. Not
surprisingly, the highest yield penalty resulted when the stress combination was applied during the
reproductive stage (Fig. 3).

Legumes and cereals display differential sensitivity to drought and heat stress combination

To develop crops with enhanced tolerance to drought and heat stress combination, a better understanding
of various strategies displayed by different crops subjected to stress combination is needed. We therefore
divided the data set into experiments that subjected cereals or legumes to a combination of drought and heat
stress (Fig. 4). Compared to drought or heat stress applied individually, both cereals and legumes were
more sensitive to the combination of drought and heat (Fig. 4A); with legumes showing a higher sensitivity

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(in yield) to drought than heat, whereas the impact of the individual stresses was not different in cereals.
To further dissect the responses of these different crop types to the stress combination, we compared relative
HI (Fig. 4B) and individual seed weight (Fig. 4C) between them. Compared to legumes, cereals had a higher
HI penalty in response to the drought and heat combination (Fig. 4B). In addition, as shown in Figs. 4C and
S3, individual seed mass of cereals was further reduced compared to legumes by the stress combination.

Impact of heat and drought stress combination on C3 and C4 crops

To investigate whether the magnified yield penalty associated with exposure to combined, as opposed to
individual, heat and drought stress is associated with C3 or C4 metabolism, we also divided the data set
based on C3 or C4 plants (Fig. 5). The vegetative tissues of C3 and C4 plants differ in responses to heat or
drought stresses, mostly due to the near abolishment of photorespiration in plants utilizing the C4
photosynthetic pathway (Crafts-Brandner and Salvucci 2002, Ripley et al. 2010, Killi et al. 2017). Although
compared to C4 plants the yield of C3 plants displayed higher sensitivity to heat, the yield of both C3 and
C4 plants was similarly impacted by the stress combination (Fig. 5).

Discussion

Global food security is influenced by many local and global factors, such as demand for food and feed,
changes in input prices (especially that of fertilizers in developing countries), soil losses due to erosion, and
water availability. Furthermore, managing the balance between conservation of farmland and natural
habitats, as urbanization increases, puts more pressure on land resources ([Link] and Lynch 2010, Seck et
al. 2012, Challinor et al. 2014). The overall economic losses and hardships resulting from episodes of
combined drought and heat wave highlight the need to study how crops respond to these events (Fischer et
al. 2005, Mittler 2006, Lobell and Gourdji 2012, Xie et al. 2018). Because the vast majority of our food and
feed are generated from field grown crops, drought and heat stress combination poses a major risk to local
and even global food security.

In crops, drought and heat combinations have a synergistic impact on life cycle, such as shortening the
number of days to anthesis and overall period required to reach maturity (Fig. 1C; Awasthi et al. 2014,

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Qaseem et al. 2019). From an evolutionary stand-point, shortening the life cycle of a plant enables it to
escape drought by reaching maturity before its occurrence becomes lethal (Prasad et al. 2008). Nevertheless,
reduced life cycle length has a profound impact on overall assimilate accumulation, overall biomass and
the length of the effective grain filling period resulting in an overall loss in seed mass in many crop species
(Fig. 4C). Because biomass accumulation is an integrator of photosynthesis rate over the growth period,
and because drought and heat combination negatively impacts both, the overall biomass of plants and the
amount of assimilates available to allocate into the grains decreases accordingly.

Yield (Figs. 1A, 3, 4A and 5) can be quantified by the integration of individual seed weight (Fig. 4C),
number of seeds in each reproductive unit (Fig. 2A), the overall number of reproductive units per plant
(Barnabás et al. 2008, Lawas et al. 2018a, Li et al. 2019, Qaseem et al. 2019), and when expressed on an
area basis, the number of plants per unit area. While both heat and drought as single stresses can negatively
impact each yield component, the examined literature indicates a shared trend among crop plants which
display a synergistic negative response to drought and heat combination (Figs. 1-5). When comparing the
effects of heat, drought and their combination between C3 and C4 crop plants for example, heat stress alone
decreased yields of C3 crops more than of C4 crops. However, when exposed to a combination of drought
and heat, both C3 and C4 crops displayed similar decreases in yield (Fig. 5). This result is somewhat
surprising given that C4 plants are considered, at least in their vegetative stages, as more drought tolerant
(Lopes et al. 2011).

While vegetative tissues are sensitive to drought and heat, especially under high light conditions, they may
recover from the impacts of stress (Ruehr et al. 2019, Balfagón et al. 2019). In contrast, seed abortion is
irreversible. Although, plants can display a considerable compensation potential and may produce fewer
but larger seeds under different stress conditions (Griffiths et al. 2015, Vonhof and Harder 1995), examples
of drought and heat stress combination resulting in partial or complete yield loss due to male sterility have
been recorded. In wheat, a crop with very short growth period, combined heat and drought during the early
reproductive stage (anthesis) can lead to male sterility (Nicolas et al. 1984). Unfortunately, only a few
studies attempted to compare the effects of similar drought and heat intensities between the reproductive
and vegetative stages (Fig. 3). An excellent example in barley (Hordeum vulgare) demonstrated that under
one week of combined stress conditions in either the vegetative or heading stages, photosynthesis rates
were similarly affected whereas a dramatic difference in the effect on yield was observed. While yield

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decrease was limited when the stress was applied during vegetative stages, yield loss of plants exposed to
combined stress during the heading stage was almost complete (Mahalingam and Bregitzer 2019).

Seed yields of all crops in this study displayed synergistic negative responses to combined drought and heat
(Figs. 1A, 3, 4A and 5). Across the many studies examined here, both individual drought and heat stress
generally caused a decrease in seed numbers and seed weight, and the impact on these traits was magnified
by the combination of heat and drought stress (Figs. 2A and 4C). Separating the overall results into cereals
and legumes, revealed a more dramatic reduction in seed weight in cereals (70%) than in legumes (50%) in
response to the stress combination (Fig. 4C). In contrast, the effect of the stress combination on legumes
was overall lower. The results presented in Fig. 4, suggest that, compared to legumes, cereals attempt to
compensate for their yield loss during drought and heat stress combination by reducing vegetative growth
and seed size.

Compare to legumes, cereals have a high starch and low protein content. Our results suggest that at least in
part, cereals display a greater decrease in HI in response to combined drought and heat then legumes (Fig.
4B). Because drought and heat combination results in low starch coupled with high protein seed content
(Fig. 2C), it is possible that seed carbohydrates to protein ratio plays a role in the response to heat and
drought combination, giving an advantage to legumes over cereals, at least when it comes to HI (Fig. 4B).

General tradeoffs between seed number and weight of individual seeds have been well documented for a
number of crop species (Griffiths et al. 2015, Vonhof and Harder 1995). In addition, the impact of stress
on seed number and seed weight is strongly influenced by the timing of the stress relative to reproductive
stages that are associated with the primary processes influencing these two parameters (Stone and Nicolas
1995). For example, seed size in wheat is most sensitive to heat during the early grain filling period and
becomes progressively more resilient throughout grain filling (Nicolas et al. 1984.). Moreover, lower seed
set under combined drought and heat makes sense from a sink-source standpoint, as fewer seeds means less
sink competition during the grain filling stages. Likewise, seeds cannot just shrink indefinitely in size and
stay viable, and therefore, as resources (assimilates) are limited under combined stress, the decreasing seed-
set is a trade-off and an adjustment mechanism to ensure the survival of few, but viable seeds (Figs. 2 and
4C).

10

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Clearly, the timing of the stress during different developmental stages is critical (Fig. 3). For example,
during flowering, pollen is particularly sensitive to high temperatures, especially when combined with
drought (Ruan et al. 2010, Jiang et al. 2019). A recent study also suggested that stigma functionality and
fertility are also sensitive under combined drought and heat in wheat plants, leading to flower abortion
(Fábián et al. 2019). Earlier work proposed that heat stress during anthesis can cause smaller or complete
absence of the embryo-sac (Sainiab et al. 1983), which can later result in a decrease in pod set and seed size
(Sainiab et al. 1983, Gross and Kigel 1994). The number of seeds per plant is a function of availability of
viable pollen that germinates, grows and leads to successful fertilization (Ruan et al. 2010). Thus, stress
during flowering can have a dominant effect on seed numbers. For example, Ji et al. (2010) as well as others
(Vonhof and Harder 1995, Yu et al. 2019) found that drought episodes early during the reproductive stage
cause pollen sterility. Additionally, heat stress is well-known to negatively impact pollen viability and
germination of pollen in different crops such as wheat, pea (Pisum sativum) and soybean (Sainiab et al.
1983, Salem et al. 2007, Jiang et al. 2019). Besides resource (carbohydrates) availability during the grain
filling stage, the final size of an individual seed is governed by the number of cell divisions and cell
expansion. Drought is known to negatively impact mitotic activity (Schuppler et al. 1998), and heat stress
was found to decrease the number of cells in soybean seeds (Tacarindua et al. 2012), suggesting that stress
induced decrease in cell division can negatively impact overall seed size (Fig. 4C).

A number of mechanisms including carbohydrate availability and metabolism have been explored and
documented to be associated with impaired reproductive success in response to heat or drought stress. For
instance, impaired sucrose metabolism has been suggested to underpin reduced pollen function and
fertilization under heat stress in chickpea (Kaushal et al. 2013). Consistent with the importance of sucrose
availability during flowering, sucrose infusion into stems of water limited maize can mitigate the effects of
stress on seed set (Boyer and Westgate 2004). The combination of drought and heat stress may magnify the
impact of each individual stress on the plant carbohydrate balance (Zhao et al. 2013). Indeed, recent studies
in rice suggest that sugar starvation in the floral organs is the underlying factor in reproductive failure of a
rice cultivar sensitive to the combination of drought and heat (Li et al. 2015, Lawas et al. 2018a, 2019).
Additionally, other researchers have shown an interactive negative effect of these stresses on the activity
of key sucrose metabolism enzymes such as sucrose synthase and invertase (Awasthi et al. 2014, Sehgal et
al. 2017). Together with the decline in photosynthesis, the decrease in sucrose metabolism under combined

11

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stress conditions may limit sucrose availability for pollen, resulting in lower pollen viability and
germination, and ultimately seed set (Figs. 2 and 4C). During the seed-filling stage, the above-mentioned
reduction in photosynthesis and leaf sucrose metabolism in response to the stress combination results in
lower carbohydrate availability for import into developing seeds (Fig. 2B). As starch is the key component
(by weight) in seeds of the major global staple crops (maize, rice and wheat; Sehgal et al. 2018), reduction
in source strength and carbon availability for starch biosynthesis during the grain filling stage will inevitably
lead to decrease in seed size and weight (Thitisaksakul et al. 2012). Recent findings suggest that in the
endosperm of developing wheat grains, drought or heat, and especially their combination decrease the
activity of key enzymes in the starch biosynthesis pathway such as starch synthase and starch branching
enzyme (Tetlow 2011, Thitisaksakul et al. 2012).

Harvest index (HI) describes the ratio between grain yield and the aboveground biomass (Erice et al. 2014).
It is a key trait tightly associated with significant yield increases of rice and wheat during the 2nd half of the
20th century. Both drought and heat stress are well known to negatively impact HI (e.g., Edmeades et al.
1999, Prasad et al. 2006), and were documented in the majority of studies examined here (Figs. 1B and
4B). As expected, based on the impact of stress combination on seed yield and yield components (Figs. 1-
5), combined heat and drought stress caused a further decline in HI (Figs. 1B and 4B), emphasizing that
combined stress conditions impact the reproductive outcome of crops more severely than their vegetative
growth. Interestingly, a striking contrast in HI under combined drought and heat conditions was found
between different crops (Figs. 1B and 4B) and the model plant Arabidopsis (Vile et al. 2012). A careful
study of biomass allocation in 10 Arabidopsis genotypes suggested that HI increases under combined
drought and heat (Vile et al. 2012). These results, which are in contrast to our findings, could suggest that
Arabidopsis may not be a suitable model plant for studies of biomass allocation during drought and heat
combination. In contrast, the model plant of C3 cereals, Brachypodium dystachion, demonstrated a similar
alteration in HI to what we observed among a diverse group of crop plants (Shaar-Moshe et al. 2017). Ruan
et al. (2010) suggested a very straightforward explanation as to why, under severe stress, the vegetative
organs stay alive while HI is altered. If the parent plant dies, no seeds will be produced, therefore, even
slight seed reproduction is better for ensuring the next generation. This is potentially why HI suffers a
significant reduction when plants are exposed to combined drought and heat (Figs. 1B and 4B).

12

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Heat, drought, and their combination influence many developmental and physiological processes in crop
plants. Whether occurring individually or in combination, the impact of these different stresses depends on
many factors, including the crop type and its growth habits (Figs. 4 and 5), as well as the stress intensity,
duration, and timing with respect to crop developmental stage (Fig. 3). Thus, it is not surprising that the
examination of studies that include control, heat, drought, and combined heat and drought stress treatments
revealed a wide range in relative impacts of all stress treatments and for all parameters explored in this
study. Nonetheless, the relative effect of the combined heat and drought stress treatment was consistently
more dramatic than the impact of drought or heat applied individually. This finding highlights the
importance of studies that specifically address heat and drought stress combination (Mittler 2006, Chen et
al. 2019, Zandalinas et al. 2017, 2020, Mazdiyasni and AghaKouchak 2015, Lobell and Gourdji 2012,
Lawas et al. 2018). Importantly, the results presented here underscore the need to focus on stress
combinations occurring during the reproductive growth stages, as the impact on yield is more severe than
when stress combination occurs during vegetative development (Fig. 3). This is especially important as to
date most studies examining molecular level mechanisms focused on vegetative tissues (e.g., Rizhsky et al.
2002, 2004, Zandalinas et al. 2017, 2020, Balfagón et al. 2019), while only limited knowledge is available
on the molecular events occurring in reproductive tissues in response to drought and heat stress (Li et al.
2015, Lawas et al. 2018b). A particular focus on the molecular responses of reproductive tissues to the
combination of drought and heat stress is warranted to identify mechanisms and avenues that can be targeted
to improve tolerance and thus alleviate the impact of stress combination on overall yield and grain quality.

Author contributions

IC and CH collected, analyzed the data, and generated the figures; IC, SIZ, FBF and RM wrote the
manuscript; SIZ, FBF and RM supervised the project.

Acknowledgements

This work was supported by funding from the National Science Foundation (IOS-1353886, MCB-
1936590, IOS-1932639) and the University of Missouri.

13

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Supporting information

Additional supporting information may be found online in the Supporting Information section at the end of
the article:

Fig. S1. Relative yield of different crops under heat stress (HS) drought stress (DS) and their combination
(HS + DS).

Fig. S2. Relative harvest index (HI) of different crops under heat stress (HS) drought stress (DS) and their
combination (HS + DS).

Fig. S3. Relative individual seed weight of different crops under heat stress (HS) drought stress (DS) and
their combination (HS + DS).

Data availability statement

The data that support the findings were derived from the literature cited through this manuscript.

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Figure legends

Fig. 1. The impact of drought and heat stress combination on relative yield, harvest index, and days to
maturity. (A) Relative yield of all crops surveyed under heat stress (HS) drought stress (DS) and their
combination (HS + DS). (B) Relative harvest index (HI) of all crops under HS, DS and HS + DS. (C)
Relative days needed to reach maturity under HS, DS and HS + DS. Results are expressed as percentage of
control conditions. Boxplots with different letters within each panel are significantly different at P > 0.05
according to one way ANOVA followed by Tukey’s test (n = 126 cases in A, n = 36 cases in B, and n = 20
cases in C).

Fig. 2. The impact of drought and heat stress combination on relative seed number and composition. (A)
Relative seed number in a reproductive unit (pot, spike, and ear) under heat stress (HS) drought stress (DS)
and their combination (HS + DS). (B) Relative seed starch, under HS, DS and HS + DS. (C) Relative seed
protein, under HS, DS and HS + DS. Results are expressed as percentage of control conditions, Boxplots
with different letters within each panel are significantly different at P > 0.05 according to one-way ANOVA
followed by Tukey’s test (n = 19 cases in A, n = 20 cases in B, and n = 16 cases in C).

Fig. 3. Comparison of the effects of heat stress (HS) drought stress (DS) and their combination (HS + DS)
on relative yield when the stress was imposed during the reproductive (n = 119) or vegetative (n = 11)
growth stages. Boxplots with different letters within each panel are significantly different at P > 0.05
according to two-way ANOVA followed by Tukey’s test.

Fig. 4. Comparison of the effects of heat stress (HS) drought stress (DS) and their combination (HS +DS)
on relative yield (A), harvest index (HI) (B) and relative seed weight (C) in crop plants that belong to the
cereal (n = 92 in A, n = 26 in B, and n = 27 in C) and legume (n = 33 in A, n = 20 in B, and n = 20 in C)
families. Boxplots with different letters within each panel are significantly different at P > 0.05 according
to two-way ANOVA followed by Tukey’s test.

Fig. 5. Comparison of the effects of heat stress (HS) drought stress (DS) and their combination (HS + DS)
on relative yield of crops belonging to the C3 (n = 93) or C4 (n = 21) photosynthetic pathway families.
Boxplots with different letters within each panel are significantly different at P > 0.05 according to two-
way ANOVA followed by Tukey’s test.

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n= 20

HS + DS
c

DS
b a

HS
90

80

70

60

50

40
100

Relative days to maturity (% of control)

C
n= 36

HS + DS
b

DS
a

HS
a

40
100

20
80

60

0
120
140

HI (% of control)
B
n= 126

HS + DS
b

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DS
a

HS
a

40

-20
100

20
80

60

0
120
A 140
Relative yield (% of control)
13993054, 2021, 1, Downloaded from [Link] by Cochrane Philippines, Wiley Online Library on [21/03/2025]. See the Terms and Conditions ([Link] on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
n= 16

HS + DS
a

DS
b

HS
b

110
140

90
150

130

120

100
160

Relative seed protein (% of control)

C
n= 20

HS + DS
b

DS
a

HS
a

90

80

70

60

50

40

30

20
110

100

Relative seed starch (% of control)

B
n= 19

HS + DS
b

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a

DS
a

HS
40
100

20
80

60
unit (% of control)

A
Relative seed number in reproductive
13993054, 2021, 1, Downloaded from [Link] by Cochrane Philippines, Wiley Online Library on [21/03/2025]. See the Terms and Conditions ([Link] on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
n= 11

HS + DS
b
Vegetative

DS
a

HS
a

HS + DS
n= 119

c
Reproductive

DS
b

HS
a

50
150

100

0
Relative yield (% of control)

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13993054, 2021, 1, Downloaded from [Link] by Cochrane Philippines, Wiley Online Library on [21/03/2025]. See the Terms and Conditions ([Link] on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
HS + DS
n= 20

b
Legume

DS
a

HS
a

HS + DS
n= 27

c
Cereal

DS
a

HS
a

40
100

20
80

60

Individual seed weight (% of control)

C

HS + DS
n= 20

bC
Legume
Legume

DS
a
A

HS
a
E

HS + DS
n= 26

c
Cereal
Cereal

DS
a

HS
a

40
100

20
80

60

0
120
140

Relative HI (% of control)
B

HS + DS
n= 33

c
Legume
Legume

DS
HS
a

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HS + DS
n= 92

c
Cereal

DS
a
Cereal

HS
a

80

60

40

20
120

100

0
Relative yield (% of control)

A
13993054, 2021, 1, Downloaded from [Link] by Cochrane Philippines, Wiley Online Library on [21/03/2025]. See the Terms and Conditions ([Link] on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
HS + DS
n= 21

c
C4

DS
HS
a

HS + DS
n= 99

c
C3
b

DS
b

HS
40
100

20
80

60
120
140

Relative yield (% of control)

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