Drought and heat stress are two major abiotic stresses that tend to co-occur in nature. Recent climate change models predict that the frequency and duration of periods of high temperatures and moisture-deficits are on the rise and can be detrimental to crop production and hence a serious threat for global food security. In this study we examined the impact of short-term heat, drought and combined heat and drought stress on four barley varieties. These stresses were applied during vegetative stage or during heading stages. The impact on root and shoot biomass as well as seed yields were analyzed. This study demonstrated that sensitivity to combined stress was generally greater than heat or drought individually, and greater when imposed at heading than at the vegetative stages. Micromalted seeds collected from plants stressed during heading showed differences in malt extract, beta-glucan content and percent soluble protein. Screening barley germplasm during heading stage is recommended to identify novel sources of tolerance to combined stress. Apart from seed yield, assessing the seed quality traits of concern for the stakeholders and/or consumers should be an integral part of breeding programs for developing new barley varieties with improved heat and drought stress tolerance.

Abbreviations

DP: diastatic power
FAN free amino nitrogen: JW Joe White system

Introduction

Meta-analysis of natural disasters that resulted in losses amounting to more than a billion dollars over the past three decades shows that both the frequency and intensity of such events are on the rise (Smith and Katz 2013). Current climate change models predict that the likelihood of facing extreme combinations of abiotic stresses are more likely than anticipated earlier (Stocker 2013). Among these co-occurring stresses, the impacts of heat and drought stress combination are the most devastating (Mittler 2006) and have become the subject of considerable research in the recent years (Mahalingam 2015, Lawas et al. 2018). Research on combined heat and drought stress in model plants (Rizhsky et al. 2004, Iyer et al. 2013) as well as in plants of agronomic importance have been reported (Simon-Sarkadi et al. 2005, Prasad et al. 2011, Zhao et al. 2016, Templer et al. 2017, Correia et al. 2018, Sehgal et al. 2018).

The majority of studies of abiotic stress responses in model plants impose stress combinations during the vegetative stages of plant growth (Rizhsky et al. 2004, Iyer et al. 2013). Though such experiments can provide information about the changes in root and shoot biomass and provide biological tissues for conducting omics-based analysis, they do not provide information on the consequences to final seed yield. There are numerous reports indicating that stress during post-reproductive stages is extremely detrimental to crop yields (Barnabas et al. 2008). In chickpea combined drought and heat stress inhibited seed filling and resulted in smaller and fewer seeds (Awasthi et al. 2014). In rice, transcriptomic and metabolic changes in floral organs in response to heat and drought stress combination revealed sugar starvation as a key factor associated with reproductive failure (Li et al. 2015). In corn, combined heat and drought stress experiments conducted under field conditions reduced yield significantly and may be correlated with the down regulation of the respiratory pathways such as the tricarboxylic acid cycle metabolites (Obata et al. 2015). In spring wheat, combined effects of heat and drought were greater than additive effects of heat or drought alone for leaf chlorophyll content, grain number and harvest index (Prasad et al. 2011). Thus, it is important to conduct more research on combined stresses in both model systems and crop plants during post-anthesis stages to collect agronomically relevant data to validate the detrimental consequences of climate extremes predicted for the future.

A concomitant pitfall in the majority of the combined stress studies is a lack of information on the impact of these stresses on the quality of the seeds produced. Since seeds serve as the main nutritional source for the majority of the population and form the primary raw materials for the food and beverage industries it is important to assess the seed quality in studies dealing with stress. In barley, drought or heat stress during grain filling stage not only reduces the yield (Savin and Nicolas 1996) but also deteriorates malt quality (Macnicol et al. 1993) and this has been attributed to high protein concentrations and low accumulation of carbohydrates (Wallwork et al. 1998, Mangelsen et al. 2011). Combined heat and drought stress led to drastic yield loss in barley, but the malting quality traits were not greatly impacted when compared to the singly applied heat or drought stress (Mahalingam 2017).

In this study, we report the impact of short-term (4–5 days) heat, drought and combined stress in four barley varieties. The stresses were imposed during vegetative stage on one set of plants. On another set of plants, these stresses were imposed during heading stage. The impact of the stresses singly and in combination were assessed based on seed yields, root and shoot biomass at maturity. In addition, we used the nanomashing technique for malting extremely small quantity of seeds (Schmitt and Budde 2011) and examined the impact of the stresses on six key malt quality traits that are routinely assessed by the malting and brewing industry.

Materials and methods

Soil mix

The soil mix for this study consisted of vermiculite, peat moss and sand (1:1:1). The three components were blended in a concrete mixer along with 25 g of slow release 15-9-12 Osmocote fertilizer (Everris International, Dublin, OH) per kilogram of mix. Three batches of the soil mix were then pooled together and mixed thoroughly manually in order to minimize differences in the individual components of the mix. Each empty pot was tared on a balance and then filled with the soil mix to a pre-determined weight to ensure that the amount of potting mix in each of the pots was consistent. Field capacity was determined by drying the soil-mix filled pots completely and then watering the pots till it started to drain out from the bottom. Based on measurements from triplicate pots, 550 ml of water was determined to be the field capacity. The pots were irrigated to 100% field capacity and following the complete drainage of the excess water, the weights of the pots were recorded. The weights of the saturated pots showed less than 1% variation.

Plant materials

Four different spring barley (Hordeum vulgare subsp. vulgare) varieties: Bowman, Conrad, Crystal and Garnet were used in this study. Bowman is a two-rowed feed barley developed for moisture-limited, rain-fed regions of the upper Midwestern United States (Franckowiak et al. 1985). Crystal and Garnet are two-rowed malting barleys developed primarily for the irrigated, high-input environments of the Intermountain West region of the United States, although they are adapted also to high-elevation, rain-fed environments that are not subject to high levels of heat and drought stress (Wesenberg et al. 1991, 2000). Conrad is a two-rowed malting variety developed by Busch Agricultural Resources that is adapted to western irrigated production (Plant Variety Protection No. 200300013). Seeds were imbibed in water for 3 h and three seeds were sown in each of 20 2.5-l pots containing the potting mix described above. There were 20 pots for each variety randomly arranged in the greenhouse to minimize any effects due to differences in the microenvironment in the greenhouse benches. Five pots of each variety were randomly selected for each treatment and control.

Initial growth conditions

Seedlings were irrigated manually once every 3 days and maintained in a greenhouse with 16 h day at 22°C and 8 h of darkness at 16°C. Relative humidity was around 40% and light intensity at the pot level was around 400 μmol m⁻² s⁻¹. Once the seedlings were 4 weeks old, drip rings were set up for each pot and plants were on auto-irrigation, twice a day, for a total of 550 ml per day. This amount of water was determined to be the field capacity of the pots. Stress treatments were imposed on one batch of plants from each variety after 4 weeks of growth (plants were at stage 25 on Zadok's scale). A second batch of plants of these four varieties was maintained in the greenhouse until heads were half emerged (plants were at stage 55 on Zadok's scale) for imposing the stress treatments.

Stress treatments

Pots with plants at the growth stages described above were moved into growth chambers for heat stress and combined heat and drought stress experiments. The growth chambers were programmed to approximate the light intensity in the greenhouse (450 μmol m⁻² s⁻¹; 16 h of light and 8 h of darkness; 40% humidity). Plants were acclimated in the growth chamber for 48 h before the imposition of the stress treatments.

Heat stress

Light was supplied from 4:00 through 20:00 h. The growth chamber was programmed to increase by 1°C every 15 min, starting at a baseline temperature of 22°C. The program was set to start at 9:00 h and by noon the temperature was up to 36°C. This temperature was maintained until 18:00 h. The chamber was also programmed for a gradual temperature ramp down of 1°C every 15 min such that the low temperature of 30°C was reached at 20:00 h coincident with the dark period. Plants were maintained in these chambers for 5 days. On the sixth day, the chamber was reprogrammed to simulate the conditions in the greenhouse. On day seven the plants were moved into the greenhouse (16/8 h day/night cycle at 22/16°C). During the heat stress regime plants were manually irrigated with 550 ml of water, the same amount as control plants in the greenhouse under auto-irrigation.

Drought stress

To impose drought stress, plants were given approximately 18% of the amount of water required for achieving field capacity, determined as described above. Hence plants subjected to drought stress were manually watered with 100 ml of water. Plants for the drought stress treatment were maintained in the greenhouse along with the control plants. Drought treatment was imposed for a period of 5 days, and on the sixth day returned to auto-irrigation with 550 ml per day.

Combined heat and drought stress treatment

For the combined heat and drought stress, plants were moved to the growth chambers as described for heat stress. The temperature regimes for the combined stress were identical to the heat stress treatment. Plants subjected to combined stress were irrigated manually with 100 ml of water. The combined stress experiments were conducted for 4 days. On the fifth and sixth day, the chamber was programmed to simulate the conditions in the greenhouse and on the seventh day the plants were moved into the greenhouse where they were irrigated with 550 ml per day until physiological maturity.

Physiological measurements

For each variety, treatment and growth stage combination, three leaves from each plant were used for measuring the physiological traits with a Li-Cor 6400 Portable Photosynthesis system (Li-Cor, Lincoln, NE). The following conditions were maintained during the measurements: leaf temperature, 22°C; CO₂ concentration, 400 μl l⁻¹; airflow, 0.1 l min⁻¹; white light illumination, 400 μmol m⁻² s⁻¹; relative humidity, 35%. Measurements were taken before the plants were moved into the growth chambers for the stress treatment and 24 h after the end of the stress treatments. Stomatal conductance, transpiration rates and net photosynthetic rates were recorded. These measurements were done on the batch of plants subjected to stress at the end of 4 weeks and also on the plants that were stressed during the heading stages.

Agronomic traits

Following short-term heat, drought and combined stress during vegetative or heading stages along with corresponding controls, plants were grown to maturity (Stage 90 on Zadok's scale) in the greenhouse under standard conditions described above. The masses of dry shoots and dry roots of each plant were measured at this stage. The dry mature heads from each plant was collected in brown bags and was later threshed using the small benchtop thresher (Model LT15; Haldrup, Poneto, IN). The seed mass was recorded individually for each plant and reported seed yield were based on the averages of five plants from each treatment.

Micromalting and malt quality analysis

Two grams of seeds from each variety and from each of the stress treatments were placed in stainless steel tea balls and used for micromalting in the Joe White (JW) system as described in Schmitt and Budde (2011). Seeds from each variety and treatment were micromalted using the JW system. Micromalting procedure was replicated twice.

The micromalted barley grains were used for analysis of four parameters associated with grain carbohydrate content. These include alpha-amylase activity, beta-glucan content, diastatic power and malt extract determined by refractive index. Two parameters associated with grain nitrogen modification were analyzed. These include free amino nitrogen (FAN) and soluble protein content. The experimental procedures for evaluating these parameters were as described earlier (Schmitt and Budde 2010, 2011). Each of the traits were analyzed twice from each technical replicate of the micromalted sample (n = 4 measurements per trait).

Statistical analysis

R-studio was used to conduct analysis of variance to determine the impact of stress during vegetative or heading stage compared with corresponding controls for four agronomic traits (yield, root biomass, shoot biomass and root:shoot ratio), three physiological traits (photosynthetic rate, stomatal conductance and transpiration) and six malting quality traits (alpha-amylase activity, beta-glucan, diastatic power, refractive index, free amino nirogen and soluble protein content) for each variety. If the anova indicated significant treatment effects, Dunnett's test was conducted to identify specific stress treatments that were significantly different from the corresponding controls (P-value < 0.05) for each variety.

Results

In the first stage of analysis, anova was conducted for each of the 13 traits to assess the impact of the stress treatments and developmental stage during which the stress was imposed. Analysis of interactions between the specific stress treatment and stage of stress application for each of the four varieties is summarized in Table 1.

Table 1. Statistical analysis of the agronomic, physiological and malting quality traits in response to heat, drought and combined heat and drought stress imposed during vegetative or heading stage in four barley varieties. AA, alpha amylase; BG, beta-glucan; D, drought; E, transpiration rate; FAN, free amino nitrogen; g_s, stomatal conductance; H, heat; HD, heat and drought; Head, short-term stress imposed during heading stage; ME, malt extract; ns, not significant; P_N, net photosynthetic rate; SP, soluble protein; Veg, short-term stress imposed during vegetative stage.

Category	Traits evaluated	Stress stage	Bowman			Conrad			Crystal			Garnet
Category	Traits evaluated	Stress stage	D	H	HD	D	H	HD	D	H	HD	D	H	HD
Physiological	g_s	Veg	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns
	g_s	Head	ns	<0.001	<0.001	<0.001	<0.001	<0.001	ns	<0.001	<0.001	<0.001	<0.001	<0.001
	E	Veg	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns
	E	Head	ns	<0.001	<0.001	<0.001	<0.001	<0.001	0.014	<0.001	<0.001	<0.001	<0.001	<0.001
	P_N	Veg	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns
	P_N	Head	0.000	0.006	<0.001	0.008	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
Agronomic	Shoot	Veg	ns	0.021	ns	ns	ns	0.011	ns	ns	ns	ns	<0.001	0.010
	Shoot	Head	0.032	0.003	ns	ns	<0.001	ns	ns	0.000	ns	0.004	<0.001	<0.001
	Root	Veg	<0.001	<0.001	0.006	0.016	ns	ns	<0.001	<0.001	<0.001	ns	<0.001	<0.001
	Root	Head	0.004	<0.001	<0.001	ns	<0.001	<0.001	0.016	<0.001	<0.001	0.010	0.007	<0.001
	Root:Shoot	Veg	<0.001	<0.001	<0.001	ns	ns	ns	<0.001	<0.001	<0.001	ns	<0.001	0.002
	Root:Shoot	Head	ns	<0.001	<0.001	ns	ns	<0.001	<0.001	<0.001	<0.001	ns	<0.001	<0.001
	Yield	Veg	0.002	0.000	<0.001	0.010	ns	0.019	0.000	ns	ns	ns	0.026	ns
	Yield	Head	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	0.031	<0.001	<0.001	<0.001	<0.001	<0.001
Malting quality	ME	Veg	ns	ns	ns	ns	ns	ns	0.000	ns	ns	ns	0.010	0.002
	ME	Head	<0.001	<0.001	<0.001	<0.001	0.022	0.020	ns	ns	ns	ns	ns	ns
	SP	Veg	ns	0.008	0.003	ns	ns	ns	ns	ns	ns	ns	ns	ns
	SP	Head	ns	ns	<0.001	<0.001	0.002	0.012	0.031	ns	ns	<0.001	<0.001	0.004
	FAN	Veg	ns	ns	ns	ns	ns	0.005	ns	ns	ns	ns	ns	ns
	FAN	Head	0.023	<0.001	<0.001	ns	0.037	<0.001	ns	ns	ns	<0.001	0.003	ns
	BG	Veg	0.019	0.038	0.049	0.000	0.009	0.001	0.044	<0.001	0.000	ns	ns	<0.001
	BG	Head	0.050	0.001	<0.001	<0.001	ns	ns	<0.001	ns	0.002	<0.001	<0.001	0.016
	AA	Veg	0.019	ns	ns							ns	ns	ns
	AA	Head	ns	ns	ns							0.008	ns	0.003
	DP	Veg	ns	ns	ns	ns	ns	ns	<0.001	ns	0.014	0.002	ns	0.000
	DP	Head	ns	0.001	ns	0.081	0.015	ns	ns	ns	ns	ns	0.003	0.000

Impact of stress on physiological parameters

Barley plants subjected to heat, drought and combined stress during the vegetative stage (approximately 4 weeks after sowing) showed transient symptoms of stress such as leaf curling, and wilting at the end of the treatment (Fig. S1, Supporting Information). One week following their return to greenhouse conditions after stress imposition, all the four varieties recovered and could not be visibly differentiated from corresponding controls. The three physiological traits – stomatal conductance, transpiration rate and net photosynthetic rate – measured 24 h after the end of the various stress treatments imposed during vegetative stage did not show any significant differences compared with their corresponding controls in the four barley varieties (Fig. 1A, C, E). In contrast, stress imposed during heading caused changes. Stomatal conductance in response to heat and combined stress during heading was significantly higher compared to corresponding control plants in all the four varieties (Fig. 1B). Drought stress lowered stomatal conductance in Conrad and Garnet, while Bowman and Crystal did not show any changes in this trait when compared to their controls. Transpiration rates mimicked the patterns observed for stomatal conductance, except that slight but significant changes were noted in response to drought stress for Bowman and Crystal (Fig. 1D).

Details are in the caption following the image — **Figure 1**
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Physiological measurements of barley varieties after the end of heat, drought and combined stress treatments. (A, C, E) Measurements of stomatal conductance, transpiration rate and photosynthetic rate, respectively, taken on plants subjected to stress during the vegetative stage, Zadok's scale 25. (B, D, F) Measurements of stomatal conductance, transpiration rate and photosynthetic rate, respectively, for plants exposed to stress during heading stage (Zadok's scale 55). Error bars represent standard deviations of the mean of 10 observations from five different plants for each treatment (n = 10). Statistically significant increases in measured traits when compared to non-stressed control plants are denoted by ‘+’ above the bars and significant decreases are denoted by ‘*’ above the bars (+,*P-value <0.05; ++, **P-value <0.001 and +++,***P-value <0.0001).

Comparison of photosynthetic rates after the end of stress treatment at heading stage indicated that Garnet was the most sensitive since there was a nearly 30% decrease in the photosynthetic rates following single and combined stress. Crystal showed significant reduction in photosynthesis rate following single stress with a nearly 50% reduction in response to combined stress. Bowman and Conrad were the most resilient with reference to photosynthetic rates (Fig. 1F). In fact, following single and combined stress the photosynthetic rates were higher in both of these varieties.

Impact of stress on root and shoot biomass

The dry shoot weights at physiological maturity in response to stress imposed at the vegetative stage of the plants of three varieties were comparable to their corresponding controls (Fig. 2A). Interestingly, Garnet showed an increase in the shoot biomass in response to heat and combined stress during vegetative stage. On the contrary, heat stress when imposed during heading stage significantly reduced shoot biomass in all the four varieties. Bowman and Garnet showed higher stem biomass in response to drought during heading while the latter was the only variety to show significant increase in shoot biomass in response to combined stress (Fig. 2B).

There were significant differences in root dry weight between the varieties, among treatments within each variety and based on time of stress imposition (Fig. 2C, D). Crystal exhibited significant decrease in root biomass in response to drought, heat and combined stress imposed during vegetative and heading stage. Root biomass in Bowman was reduced in response to heat and combined stress applied during vegetative or heading stage. But the root biomass showed opposite responses to drought depending on the stage of stress imposition. In Conrad the root biomass changes were significantly increased in response to combined stress while it was decreased by heat stress during heading. Interestingly, Garnet showed a significant increase in root biomass in response to heat stress during vegetative stage while this pattern was reversed when the heat was imposed during heading. Combined stress led to an increase in root biomass in Garnet, regardless of the stage when the stressor was applied.

Crystal showed the most significant reduction in the root:shoot ratio in response to single and combined stress during vegetative or heading stages (Fig. 2E, F). With the exception for the drought stress during heading, significant reduction in root:shoot ratio was observed for Bowman. Conrad had the least amount of variation in root:shoot ratio except for the significant increase in this trait in response to combined stress during heading. Garnet was the only variety to show a significant increase in root:shoot ratio in response to combined stress and in response to heat stress during vegetative stage.

Impact of stress on seed yields

Singly applied drought or heat and combined stress during vegetative stage led to significant reduction in seed yield in Bowman (Fig. 2G). Garnet showed a slight reduction in yield in response to heat stress during vegetative stage but was not affected by drought or combined stress. Seed yields of Crystal were nearly doubled in response to drought stress during vegetative stage. Heat and combined stress during vegetative stage did not cause any significant effect on seed yields of Crystal. Similar to Crystal, Conrad showed significant increase in seed yield in response to drought but combined stress led to a significant reduction. There was a dramatic reduction in seed yield in all the four lines when the stresses were imposed during heading stage (Figs 2H and S2). Overall, yield loss in response to drought stress was less severe compared to heat stress while the combined stress led to more than 95% reduction in the yields of all the four tested varieties. Crystal was the most drought-tolerant variety since the yield loss was minimal when the stress was applied during heading. Based on the seed yield analysis, Crystal seems to be the most suitable for overcoming short-term drought during vegetative or heading stage. None of the four varieties will be apt when facing heat or combined stress scenario during heading stage.

Impact of stress on malting quality traits

Malt extract

Imposing stress during vegetative stage made the least impact on malt extract of Bowman and Conrad. Interestingly, malt extract percent increased in response to drought in Crystal (Fig. 3A). This was observed in Garnet in response to heat and combined stress. However, when the stress was imposed singly or in combination during heading, Bowman and Conrad showed significant reduction in malt extract. Malt extract was not compromised in response to stress during heading stage in Crystal and Garnet (Fig. 3B).

Soluble protein

Imposing stress singly or in combination during vegetative stage did not affect percent soluble protein in Conrad, Crystal and Garnet (Fig. 3C). However, in Bowman variety soluble protein increased in response to heat during vegetative stage and combined stress during vegetative or heading stage. In Garnet, stress during heading stage led to a significant increase in soluble protein (Fig. 3D). In Crystal only drought stress during heading led to slight reduction in the soluble protein. Interestingly, in Conrad drought and combined stress during heading led to an increase in soluble protein while heat stress caused a significant decrease.

Free amino nitrogen

With the exception of combined stress in Conrad none of the other varieties showed any significant change in FAN in response to single or combined stress imposed during vegetative stage (Fig. 3E). Significant increase in FAN was observed in response to heat during heading in Bowman, Conrad and Garnet. This pattern was also observed for combined stress in Bowman and Conrad. Drought during heading significantly increased FAN levels in Garnet while in Bowman it caused a decrease (Fig. 3F). FAN levels in Crystal were unperturbed in response to single and combined stress during heading.

Beta-glucan

Levels of beta-glucan were the most variable of all the malting quality traits and changes were observed in response to stress imposed in vegetative or heading stage (Fig. 3G, H). The changes were observed within each variety and among the stresses. In Conrad, levels of beta-glucan increased in response to single and combined stress during vegetative stage, while this was observed only in response to drought stress imposed during heading. In Crystal, beta-glucan levels increased in response to drought and combined stress during vegetative and heading stages. Significant increase in beta glucan was observed in response to heat stress imposed during vegetative stage in Crystal that was not seen when the stress was imposed during heading. In Garnet, levels of beta-glucan were reduced in response to single and combined stress imposed during heading, while this was true only for combined stress applied during vegetative stage. In Bowman, drought during vegetative and heading stage increased levels of beta-glucan significantly while their levels plummeted in response to combined stress (Fig. 3G, H).

Alpha amylase

In Bowman, drought stress during vegetative stage led to a decrease in alpha-amylase activity (Fig. 3I). A significant increase in amylase was observed in Garnet in response to drought and combined stress during heading stage (Fig. 3J). No significant treatment or stage of stress imposition was observed with reference to alpha-amylase activities in Conrad and Crystal (Table 1).

Diastatic power

A significant reduction in diastatic power was observed in Crystal and Garnet in response to drought stress during the vegetative stage (Fig. 3K). Heat stress during heading increased the diastatic power in Bowman, Conrad and Garnet (Fig. 3L). While combined stress during vegetative stage increased diastatic power in Crystal, it significantly reduced diastatic power (DP) in Garnet. Single and combined stress during vegetative stage did not affect DP in Bowman and Conrad and this pattern was observed for Crystal when the stresses were imposed during heading (Fig. 3K, L).

Discussion

The majority of the barley in the United States is used by the malting and brewing industry with lesser amounts being used for food and feed. For malting, two-rowed spring types predominate hence we decided to focus on these in this study.

Physiological responses to short-term drought, heat and combined stress vary depending on the developmental stage during which stress is imposed

The overall phenotypes of the barley varieties were relatively less sensitive to short-term heat or drought or combined stress during the vegetative stage (Fig. S1). Within 1-week following the stress treatments the plants looked visually healthy and in fact some of the plants were more robust than their controls (data not shown). Physiological measurements of photosynthesis, transpiration and respiration were similar in the post-stress plants and controls. Thus, based on visual symptoms we could suggest that all these four lines are tolerant to heat, drought and combined stress. However, the responses to heat, drought and combined stress during heading stage evoked a very different physiological response in each of the four varieties (Fig. 1). Contrary to expectations, Bowman and Crystal did not lower their stomatal conductance in response to drought stress and may be worthy of further investigation. All four varieties showed higher stomatal conductance in response to heat stress. The higher stomatal conductance observed in response to combined stress in all four varieties suggests that barley in general may be more sensitive to heat than drought. Similar observations have been made based on a survey of barley varieties grown in the United States (Mahalingam 2017) and Europe (Peltonen-Sainio et al. 2010, Hakala et al. 2012). A significant reduction in the photosynthetic rates in response to combined stress in Crystal and Garnet is consistent with a previous report using a Syrian barley landrace Arta and Australian variety Keel (Rollins et al. 2013). However, the higher photosynthetic rate observed in Bowman and Conrad in response to single and combined stress suggests variation for this trait in extant germplasm.

Short-term combined drought and heat stress during heading is detrimental to barley yield

Analysis of agronomic characteristics such as shoot and root biomass show that heat stress applied during heading took a heavy toll on all the four lines (Table 1, Fig. 2). Based on this observation we speculate that these varieties in general may lack the genetic mechanisms for overcoming heat stress during heading. Shoot biomass did not show changes in response to drought stress during vegetative stage in all the varieties, but this pattern was not observed when the stress was imposed during heading. Thus, depending on the stage of the plant during which the stress is imposed, the same variety(ies) can show completely contrasting results. This was also reported in another study using wild barley lines (Zhao et al. 2010, Wu et al. 2015). Hence in screening studies for identifying new sources of tolerance to abiotic stresses it is extremely important to consider the impact of phenology during which the stressors are evaluated.

Root/shoot ratios could be used to describe the carbon source and sink balance in plants (Gleeson 1993). In Crystal plants wherein, the root growth was compromised due to stress during vegetative stage, shoot growth was not altered, and the final seed yield was generally comparable to controls or higher than controls (drought). This suggests that varieties with a reduced root and high shoot biomass, in other words low root:shoot ratio, could aid in enhancing sink balance which could further sustain seed yield during drought and heat stress. However, these observations could not be extended when the stress was imposed during heading stage. This indicates that screening for abiotic stress tolerance should take into consideration not only the phenology during which a stress-related trait is evaluated but also the impact of the measured traits on the final seed yield.

If we consider only the seed yield data for assessing the performance of these four varieties in response to single and combined heat and drought stress, again the conclusions will be different depending on whether the stress was imposed during vegetative or heading stage. With the exception of Bowman, the other three varieties were comparable to control when the stress was imposed during vegetative stage (Fig. 2). The higher yields recorded for Conrad and Crystal subjected to drought is indeed worthy of further exploration using molecular biological approaches. At a whole plant level, these droughted plants were more robust compared to their corresponding controls suggesting some innate mechanism (maybe hormonal) that triggers their ability to thrive well following a short-term water stress. The lower reduction in seed yields in response to drought during heading compared to heat and combined stress indicates that these varieties have tolerance to water deficit and could be attributed to physiological responses consistent with drought-tolerant plants in general (Fig. 1). The cultivars chosen for this study are adapted best to regions with relatively cool climate where the maximum temperatures during the heading stages are usually between 26 and 29°C. The US varieties bred for these temperate to dry sub-tropical climes are in general capable of handling water-deficit but are negatively impacted when the temperature increases by 5–6°C more than average temperature even for a few hours and a short period of 5 days. This is further reflected in the higher stomatal conductance and transpiration rates in all the four varieties, an expected response to heat stress but also in response to combined stress (Fig. 1). It has been reported that abiotic stress during post-anthesis stages is extremely detrimental to plants in general and cereals in particular (Barnabas et al. 2008). The combined heat and drought stress were the most severe resulting in more than 95% loss in yield in all the four varieties. In a larger survey using 18 different US barley malting varieties, we observed a similar trend in response to combined heat and drought stress applied during heading stage (Mahalingam 2017). This study again emphasizes the extreme vulnerability of US barley production to higher temperatures predicted for the future, which can be further exacerbated by water-deficits.

Short-term drought, heat and combined drought and heat stress imposed during heading affect key malting quality traits in barley varieties

Finally, we sought to examine the impact of abiotic stress on the malting quality (Fig. 3). Five of the six traits examined did not show any significant changes when the stresses were imposed during vegetative stage in majority of the varieties. Beta-glucan was the only malting quality parameter that showed extensive variation whether the stress was imposed during vegetative or heading stage (Fig. 3). It is noted that the measurements of beta-glucan can be impacted by the tea ball micromalting procedure used in this study compared to the traditional malting protocols (Schmitt and Budde 2011). Beta-glucan is a polysaccharide that affects malt quality since residual beta-glucan in malt will increase wort viscosity leading to difficulties in downstream filtration process and cause chill haze in beer (Mcclear and Glennieholmes 1985). Extensive variation in beta-glucan levels have been reported in barley genotypes (Molinacano et al. 1989) and variation in beta-glucan has been reported in response to environmental and agronomic factors (Wang et al. 2004). The lowering of beta-glucan levels in response to single and combined stress in variety Garnet is particularly interesting. It has been reported that stress-induced thaumatin-like proteins bind to beta-glucans and facilitate their removal during malting (Singh et al. 2017). It is tempting to speculate that an increase in protein levels in Garnet may be contributing to the lowering of the beta-glucan levels in this variety. Analysis of the expression of thaumatin-like proteins in these barley varieties can lend credence to these speculations and provide a novel avenue for improving malting quality.

Total protein content of barley seeds can cut both ways in determining malt quality. High protein will lower carbohydrate content and hence malt quality and beer. Low protein content in the grains can reduce the activities of starch-degrading enzymes, reduced foam formation and beer palatability. An increase in seed protein content and in turn beta-amylase activity has been reported in response to water deficit in barley (Wu et al. 2015). Consistent with this study, higher protein levels in variety Garnet did correlate with higher alpha-amylase activity in response to drought and combined stress, and higher diastatic power in response to heat. However, this trend was reversed in Conrad with lower protein levels in response to heat but higher diastatic power. This indicates that there are varietal differences even in the currently grown varieties that can be exploited for breeding barley tolerant to stresses with lower protein and higher enzyme activities.

Conclusions

We show that barley varieties subjected to short-term abiotic stress combinations during vegetative stages can thrive well later and their seed yields and quality are comparable to non-stressed plants. On the contrary, plants subjected to combined heat and drought stress during heading stages are heavily compromised in terms of seed yield and show varietal specific changes in malting trait profiles. Based on this study and our previous study with 18 US barley malting varieties (Mahalingam 2017) we conclude that extant barley varieties are highly vulnerable to episodic regimes of heat stress and combined heat and drought stress during heading. Further, this study supports the urgent need for screening barley germplasm collection to identify lines with tolerance to heat and drought stress during heading that can then be used in breeding programs for enhancing adaptive capacity of new varieties that can thrive well under the predicted climate change scenarios for the future. A comprehensive analysis of the transcriptome, proteome and metabolome of the barley lines with tolerance to combined stress can provide novel markers for marker-assisted selection and molecular breeding in the future.

Author contributions

R.M. and P.B. designed this research. P.B. provided all the seed materials for conducting this research project and editing the manuscript. R.M. conducted the research, analyzed the data and wrote the manuscript.

Acknowledgements

The authors thank Danielle Graham for growing the plants, her technical assistance in conducting the stress experiments and micromalting. The authors thank Dr Jason Walling and the Cereal Crops Research Unit Malting Facility for access to the Joe White system for micromalting.

Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.

Supporting Information

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Citing Literature

Volume165, Issue2

Special Issue:Stress Combination

February 2019

Pages 277-289

This article also appears in:

Drought

Impact on physiology and malting quality of barley exposed to heat, drought and their combination during different growth stages under controlled environment

Abstract

Abbreviations

Introduction

Materials and methods

Soil mix

Plant materials

Initial growth conditions

Stress treatments

Heat stress

Drought stress

Combined heat and drought stress treatment

Physiological measurements

Agronomic traits

Micromalting and malt quality analysis

Statistical analysis

Results

Impact of stress on physiological parameters

Impact of stress on root and shoot biomass

Impact of stress on seed yields

Impact of stress on malting quality traits

Malt extract

Soluble protein

Free amino nitrogen

Beta-glucan

Alpha amylase

Diastatic power

Discussion

Physiological responses to short-term drought, heat and combined stress vary depending on the developmental stage during which stress is imposed

Short-term combined drought and heat stress during heading is detrimental to barley yield

Short-term drought, heat and combined drought and heat stress imposed during heading affect key malting quality traits in barley varieties

Conclusions

Author contributions

Acknowledgements

Supporting Information

References

Citing Literature

Figures

References

Related

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