Generating Plants with Improved Water Use Efficiency

Abstract

To improve sustainability of agriculture, high yielding crop varieties with improved water use efficiency (WUE) are needed. Despite the feasibility of assessing WUE using different measurement techniques, breeding for WUE and high yield is a major challenge. Factors influencing the trait under field conditions are complex, including different scenarios of water availability. Plants with C₃ photosynthesis are able to moderately increase WUE by restricting transpiration, resulting in higher intrinsic WUE (iWUE) at the leaf level. However, reduced CO₂ uptake negatively influences photosynthesis and possibly growth and yield as well. The negative correlation of growth and WUE could be partly disconnected in model plant species with implications for crops. In this paper, we discuss recent insights obtained for Arabidopsis thaliana (L.) and the potential to translate the findings to C₃ and C₄ crops. Our data on Zea mays (L.) lines subjected to progressive drought show that there is potential for improvements in WUE of the maize line B73 at the whole plant level (WUE_plant). However, changes in iWUE of B73 and Arabidopsis reduced the assimilation rate relatively more in maize. The trade-off observed in the C₄ crop possibly limits the effectiveness of approaches aimed at improving iWUE but not necessarily efforts to improve WUE_plant.

1. Introduction

Green Revolution technologies and significant expansion in the use of land, water, and other natural resources for agricultural purposes have led to a tripling in agricultural production between 1960 and 2015 [1]. Despite this success, the high costs to the natural environment that accompany elevated productivity and changes in the food supply chain threaten the sustainability of food production [1]. Global food security is further challenged by climate change, with a predicted increase in frequency of droughts [2,3]. Globally, agriculture accounts for at least 70% of withdrawals from freshwater resources, with large effects on ecosystems [4,5]. Despite this high water deployment, major yield losses due to water deficits are experienced in crops [6]. At the same time, global population growth increases the demand for food, feed, and fuel, which intensifies the pressure to improve water use efficiency (WUE) of crops [7,8]. While better crop and water management practices provide an immediate opportunity to increase crop water productivity, breeding for superior varieties can achieve a medium- and long-term increase [9,10].

Physiologically, water use efficiency can be defined at different scales [11,12,13]. At the plot level, it represents the ratio of grain or biomass yield to water received or evapotranspired. At the single plant level (WUE_plant), it is the ratio of biomass to transpiration. The increase in biomass and amount of water transpired over time can be assessed gravimetrically [14]. However, this is destructive and laborious on a long-term basis, especially regarding large crops like maize and sorghum. Therefore, analyses of intrinsic water use efficiency (iWUE) and carbon isotope discrimination (Δ¹³C) are used as surrogates when evaluating WUE [11,15]. The iWUE is assessed at the leaf level as the ratio of net CO₂ assimilation (A_n) to stomatal conductance (g_s) and can be measured noninvasively with portable gas exchange equipment [16]. As transpiration rate (E) is influenced not only by g_s but also by the leaf-to-air vapor pressure deficit (VPD) of the air [17], iWUE usually differs from transpiration efficiency (A_n/E). In addition, VPD affects the stomatal aperture and therefore g_s [17,18]. Extrapolation of gas exchange data from single-leaf to whole plant is error-prone due to differences in photosynthesis and transpiration among leaves [19]. Prediction of long-term biomass accumulation and water consumption, WUE_plant, based on iWUE is even more uncertain given the possible differences in VPD and additional physiological processes such as dark respiration and photorespiration influencing the resulting biomass increase [19]. Despite these limitations, analysis of iWUE provides a convenient measure for the water efficiency of carbon capture. The throughput of iWUE analyses is quite low as only single, time-consuming measurements per plant can be taken, which impedes large-scale phenotyping.

Analysis of stable carbon isotope discrimination (Δ¹³C) offers a suitable alternative in C₃ plants by providing a read-out for transpiration efficiency integrated over time. Discrimination of the heavier isotope is mainly caused by differences in diffusion rates of the isotopes and enzymatic discrimination during carboxylation reactions [20]. Therefore, Δ¹³C has been used as an indirect trait to select cultivars with improved WUE [21,22,23,24]. By combining the analysis with oxygen isotope enrichment Δ¹⁸O, an estimation for transpiration rate [25,26,27], contributions of water loss, and CO₂ assimilation on iWUE could be disentangled [28,29,30]. Stable isotope compositions of leaves or grains, however, represent integrated measures of many processes over a period of plant growth and therefore correlation with iWUE can be limited [31]. In C₄ plants, CO₂ prefixation, for instance, by phosphoenolpyruvat carboxylase and bundle sheath leakiness restrict the responsiveness of Δ¹³C to changes in WUE [20,32] and make the relationship between Δ¹³C, g_s and WUE in C₄ species less predictable compared to C₃ plants [13].

Improving WUE of crops is considered beneficial in very dry climates and in very severe and terminal drought conditions, while growth maintenance traits are advantageous under milder drought conditions [33,34,35]. For crops experiencing water deficit early in their development, traits found to be positive for improving WUE are negative for yield [36]. Enhanced water uptake through investments in the root system can result in reduced plant size and water expenditure for growth maintenance can result in increased drought stress experiences if plants are growing at very low soil water availability [33,34,36,37]. Hence, water-conserving traits as imposed by higher WUE would be beneficial, provided growth and yield are not negatively affected.

2. Disconnecting Improved WUE and Growth Trade-Offs

Being a ratio, iWUE can be improved by reducing g_s per amount of CO₂ assimilated or by enhancing the assimilation rate at a given g_s. Both cases result in lowered intercellular CO₂ concentration (C_i) and consequently in an increased stomatal CO₂ gradient (C_a − C_i, with external CO₂ concentration C_a), which is directly proportional to the ratio of A_n to g_s according to Fick’s law applied to carbon assimilation in leaves, A_n = g_s (C_a − C_i) [16,38,39]. Increased iWUE has been observed in several C₃ species under water deficit conditions when plants reduce g_s [11,15,40,41,42], although a decrease in g_s caused by drought was found to be overridden by heat stress [43]. However, closing stomatal pores to reduce transpiration often results in a reduction of A_n [41,44]. Lowering g_s impinges on C_i and unless this change in C_i is counteracted by an elevated mesophyll conductance (g_m), the CO₂ concentration at the site of Rubisco-dependent carboxylation (C_c, CO₂ concentration in chloroplasts) will be reduced [39,45,46]. A reduction of C_c affects the carboxylation efficiency of Rubisco and favors photorespiration [47,48]. Sustaining net photosynthesis under these conditions might require a higher electron transfer rate (ETR) and/or reduced nonphotochemical quenching to support enhanced carboxylation by Rubisco for compensation of enhanced photorespiration [49,50,51,52]. There are reports that water deficit results in increased g_m; however, in most analyses, no change or a reduced g_m was observed under drought [44,53,54,55,56,57,58].

Gains in WUE are often associated with growth trade-offs [59,60]. As pointed out by Blum [61], crops with high CO₂ assimilation and high biomass accumulation per unit land area require high stomatal conductance. This is supported by the observation of a constant WUE on the field level over a broad range of yields [8]. Nevertheless, there might be ways to achieve elevated WUE and high photosynthesis, namely by exploring CO₂ concentrating mechanisms, increased g_m, and increased CO₂ specificity of Rubisco [10].

Interestingly, several reports of C₃ plants have shown enhanced iWUE without the expected negative impact on A_n or growth [11,15,62,63,64], as postulated by plant physiologists [4]. In these studies with transgenic tomato and Arabidopsis plants, g_s was moderately reduced by enhancing the biosynthesis or the responsiveness to the phytohormone abscisic acid (ABA) or by reducing the size and density of leaf stomata [15,63,64,65]. Plants overexpressing distinct ABA receptors—termed ABA-Binding Regulatory Component (RCAR)/Pyrabactin Resistance 1-(like) (PYR1/PYL)—caused increases of 40% in iWUE, integrated WUE based on Δ¹³C of biomass and cellulose fractions, and WUE_plant [15]. Growth rates and biomass accumulation were not significantly different from wild type [15]. Hence, the ABA receptor lines revealed higher water productivity, i.e., WUE per time, both under well-watered growth conditions and under water deficit. Net carbon assimilation was comparable to the wild type, however, at lowered C_i levels and without detectable changes in g_m. This report and other studies show that improving WUE is possible without growth trade-offs. The underlying physiological mechanisms are largely unknown and might involve the root system, as grafting experiments have suggested [15], and enzymes of the C₄ metabolism, such as PEP carboxylase and its regulatory protein kinase PEPC kinase, which are both upregulated in C₃ plants at low CO₂ availability [65].

C₄ and C₃ plants differ in WUE [66,67,68]. At a given g_s, C₄ plants show higher net carbon assimilation rates and higher WUE [66]. The CO₂ concentrating mechanism involving PEP carboxylase results in saturation of C₄ photosynthesis at relatively low C_i [69,70]; therefore, lower g_s and a steeper CO₂ gradient (C_a − C_i) are realized in C₄ plants compared to C₃ plants [66,70]. C₃ plants have C_i values in the range of 300 ± 60 µmol mol⁻¹, while the C_i of the C₄ plants is around 150 ± 40 µmol mol⁻¹ [71,72,73] at ambient CO₂ of 370–400 µmol mol⁻¹ in well-watered conditions. Under optimal growth conditions, maize and sorghum with C₄ metabolism therefore have higher yields per water transpired than the C₃ crop wheat [9].

3. Comparative Analysis of Maize and Arabidopsis

The question arises as to whether it is possible to transfer the finding of improved iWUE without having the negative impact on growth to crops. The data on g_s-modified tomato plants suggests that it might work for C₃ plants [63], but the lower C_i level of C₄ plants could preclude such an accomplishment in maize.

To explore the relevance of these findings of uncoupling WUE improvement and yield decreases for the C₄ crop maize (Zea mays L.), we analyzed gas exchange data obtained from the maize inbred line B73 and compared them to findings in Arabidopsis. In addition, we analyzed the WUE_plant of maize lines subjected to drought. B73 is an inbred line that is commonly used in breeding programs, but is known to be drought-sensitive [74]. B73 was included in a progressive drought stress experiment adapted from Yang et al. [15] in which biomass production with a given amount of water was analyzed and WUE_plant was determined. In this experiment, B73 showed the lowest WUE_plant (Figure 1a) compared to the maize inbred Mo17 and lines derived from an introgression library described by Gresset et al. [75]. In Figure 1, data are shown of the recurrent parent (RP) of the introgression library as well as two introgression lines differing from RP by reduced (IL-05) or elevated kernel Δ¹³C (IL-81) [75]. A significantly reduced WUE_plant compared with the recurrent parent for IL-05 shows the potential of genetic improvement for this trait. However, the largest difference in WUE_plant was observed between B73 and Mo17, with an increase of ~27% (Figure 1a). Our data is in accordance with a previous drought stress experiment conducted on seedlings of maize inbred lines, where Mo17 ranked top in yield per plant [76]. The results indicate genetic variation in the efficiencies of water use among maize lines and a potential for genetic improvement of the WUE_plant for B73.

Maize lines showed a difference in water consumption over the five weeks of the experiment (ANOVA, p < 0.001, Figure 1b). However, differences in water consumption cannot explain the differences observed in WUE_plant and towards the end of the progressive drought, all genotypes included in the experiment had consumed an equal amount of water (5.8 kg ± 0.02 kg, mean ± SE).

The way in which the change in soil water content (SWC) during the progressive drought experiment affected photosynthesis and iWUE was analyzed by gas exchange measurements. The A_n of leaves was fairly constant for maize B73 plants exposed to high SWC levels up to 40%, then the A_n dropped steadily approaching zero at approximately 20% SWC (Figure 2a). In parallel, g_s changed moderately between 70% and 40% SWC and declined to zero at 20% SWC (Figure 2b). The C_i values were in the range of 80–100 µmol CO₂ per mol between 40–60% SWC. They were somewhat higher in plants from water-saturated soil and were lowered to a minimum of approximately 40 µmol mol⁻¹ at 25% SWC (Figure 2c). Further reduction of the water content in the soil resulted in the steep rise of C_i values, indicating collapsing photosynthesis at very low g_s of plants experiencing severe drought stress. As the ambient CO₂ concentration (C_a) surrounding the leaf was maintained at 400 µmol mol⁻¹, the CO₂ gradient (C_a – C_i) at the stomatal pores increased from approximately 250 µmol mol⁻¹ (C_i of 150 µmol mol⁻¹) at soil water saturation to approximately 360 µmol mol⁻¹ (C_i of 40 µmol mol⁻¹) at the brink of terminal drought. The SWC also influenced iWUE (Figure 2d). Values increased from well-watered conditions to a maximum at 25% SWC, with a plateau around 170 µmol CO₂ per mol H₂O between 40–60% SWC. Under mild water deficit between 40–60% SWC, there was little variation in A_n, and g_s and, consequently, the iWUE values.

The results for maize B73 differed from data gained by similar analyses of Arabidopsis plants (Figure 3a–d; Reference [15]). The A_n remained constant between 30–70% SWC, which might be caused by light-limited, but not water-limited, photosynthesis. However, g_s and C_i steadily decreased with decreasing SWC and, concomitantly, the iWUE increased by twofold from approximately 35 to 70 µmol mol⁻¹ at 30% SWC. The CO₂ gradient at stomata increased more than twofold from approximately 80 µmol mol⁻¹ at soil water saturation to approximately 170 µmol mol⁻¹ at 30%. The data were obtained at light conditions that did not saturate photosynthesis, but analysis at saturating light confirmed the capacity of Arabidopsis to lower C_i and maintain photosynthetic rates unchanged [15]. The improvement in iWUE by limiting g_s without major trade-offs in A_n (Figure 3) was observed for the C₃ plants Arabidopsis [15,24] and tomato [63]. A twofold enhancement in iWUE has been reported in different C₃ species under drought [11,15,77,78,79]. Besides, considerable differences in WUE in the absence of drought stress have been observed among natural variants [80,81].

Comparing the results from the analysis of maize and Arabidopsis, the potential for increasing iWUE in maize was more limited relative to Arabidopsis. Between 30–60% SWC—reflecting mainly mild water deficit [15]—Arabidopsis responded to the mounting water deficit by a 70% increase in iWUE, while maize showed an increase of less than 20%. Based on the iWUE values obtained at water-saturated soil, the iWUE increase in Arabidopsis and maize was approximately 100% and 40%, respectively, but water logging might be an issue at these high SWC levels. Between 30% and 60% SWC, the stomatal CO₂ gradient of maize increased from 300 to 350 µmol mol⁻¹, which corresponded to a 17% increase, whereas in Arabidopsis, the gradient was enhanced by 70%, from 100 to 170 µmol mol⁻¹.

To sum up, mild drought stress (30–60% SWC) had a minor effect on A_n, g_s, and iWUE in maize. However, in Arabidopsis, g_s and iWUE changed dynamically, while A_n was little affected. A reduction in SWC from 35% to 25% led to a rapid decline of A_n in maize.

These results are in accordance with data from C₃ and C₄ grass species [77,78] but only partly meet the behavior expected for C₃ and C₄ plants based on a meta study [82] where decreases in g_s and A_n under mild drought stress were more pronounced in C₃ relative to C₄ species. Comparing the A_n and g_s curves of maize B73 and Arabidopsis Col-0 (Figure 2a,b and Figure 3a,b), a reduction in stomatal conductance led to an immediate reduction in assimilation rate A_n for maize but not for Arabidopsis. However, not all C₃ plants show the same flat A_n/g_s curve as Arabidopsis [83,84] and therefore the data cannot be translated to C₃ crops in general. A previous study on maize lines found differences in g_s without trade-offs in A_n at well-watered conditions [85], and the author noted that C_i values did not become low enough to limit A_n as it might in high VPD conditions [85] or as it was observed here under drought.

Still, the limitations in improving WUE in maize B73 could be unique to this inbred line, and comparable data from other maize lines is needed before implications can be expanded to maize in general. Additionally, results from maize cannot represent C₄ photosynthesis in general because this pathway evolved independently in 19 angiosperm families [67]. However, other C₄ grass species have also shown a slight increase in iWUE with progressive depletion of water followed by a steep decline under very severe drought conditions driven by a pronounced decline in A_n [77,78]. The loss of A_n in the C₄ grass species under drought could partly be attributed to stomatal limitations, while other limitations dominate, including photoinhibition, limitations of CO₂ fixation due to desiccation, and decreases in g_m [73,77].

It has also been shown that for subspecies of Alloteropsis semialata (R.Br.) Hitchc., A_n is massively reduced in C₄ subspecies under drought in such a way that C₄ photosynthesis totally loses its advantage over photosynthesis of the C₃ subspecies [73]. This conclusion cannot be drawn from our experiments. However, the observation that C₃ plants become more water use efficient during mild and moderate drought while C₄ plants show more stable WUE [86] is in accordance with our data on iWUE.

The less potent improvement in WUE observed in maize under drought might be attributable to a limitation in increasing the CO₂ gradient (C_a − C_i) further. Maize, like other C₄ species, possesses a CO₂ concentrating mechanism utilizing precarboxylation of CO₂ by PEP carboxylase, which results in C_i values approximately half compared to C₃ species [87]. Such a mechanism results in advantages in A_n and WUE under non- or mild-water-deficit conditions [9,66,69,73,77,78]. However, these advantages cannot be maintained when the drought gets severe, especially for maize and C₄ grass species [66,69,73,77,78,86], which is in agreement with our observation for maize B73 at SWC below 27%. The differences can also translate to the field level, where maize has been found to be more sensitive to drought than wheat (C₃), with yield reductions of 39.3% compared to 20.6%, respectively, at approximately 40% water reduction [88]. Maize and sorghum are equally or even more sensitive to water stress than many C₃ plants [69,73].

Our results show a potential to increase WUE in maize. The inbred lines displayed a broad variation in WUE_plant under progressive drought, and iWUE—measured under the same conditions for the least efficient line at whole plant level—still showed a moderate increase in iWUE with declining SWC. However, the potential of WUE improvement is limited in this C₄ plant compared to Arabidopsis. This limitation is caused by a very high iWUE and low C_i under well-watered conditions, which provides a minor degree of freedom for further lowering the C_i. The C₃ plant is more responsive concerning increases in iWUE under mild water deficit compared to maize. Hence, screening C₃ plants for enhanced iWUE in combination with efficient growth is a suitable approach to identify crops with improved WUE_plant. This approach is less promising for C₄ plants. Establishing higher C_a − C_i gradients in C₃ crops at a given soil water potential, e.g., by biotechnical engineering using ABA receptors, has the potential to increase iWUE at the cost of minor reductions in A_n. Moderate reductions in A_n do not necessarily influence yield. In barley, improvements in iWUE and WUE_plant have been associated with trade-offs in carbon assimilation but without deleterious effects on plant growth or seed yield [89].

4. Conclusions

Our results indicate that the improvement in iWUE without trade-offs in carbon assimilation, as observed for tomato and Arabidopsis [15,63], is less promising for maize and possibly other C₄ plants. The large CO₂ gradient established by the CO₂-concentrating mechanisms of C₄ plants limits the potential for further increases in iWUE compared to C₃ plants. However, our data show major differences in WUE_plant for maize inbred lines and therefore potential for genetic improvement of this trait.

A recent meta-analysis on WUE revealed a tenfold bias in favor of C₃ plant studies compared to analyses on C₄ plants [86]. We therefore see an urgent need for more studies on C₄ crops to shed light on the mechanisms of WUE under water deficit in these important but drought-sensitive crops. Cereals like rice, maize, and wheat contribute largely to global food security [90]; therefore, breeding for and generating water-efficient and high yielding crops are an urgent task to meet future challenges.