Roots Withstanding their Environment: Exploiting Root System Architecture Responses to Abiotic Stress to Improve Crop Tolerance

1The world population is growing rapidly and this is accompanied by an increased food demand. In past decades, this growing food demand has been addressed by plant breeding consistent with optimal conditions for plant growth. In agricultural practices, the use of fertilizers, irrigation, pesticides, and other inputs can create these optimal conditions on the short-term. However, increasing evidence exists for the negative consequences of these practices on the long-term.

2First of all, irrigation accounts for almost 70% of all freshwater usage in the world. Freshwater scarcity is a big threat to the human population and the current water usage for agriculture is not sustainable. Furthermore, irrigation causes salinization of soils and increases leaching of fertilizer. This leaching, together with excess use of fertilizer and deep tilling leads to higher greenhouse gas emissions.

3These problems illustrate the unsustainability of creating the optimal conditions our crops are selected for. In addition, climate change will further increase this challenge. Agriculture will have to deal with growing crops under suboptimal conditions, creating a gap between the yield potential and the currently reached yield – the so-called yield gap. An extensive research field tries to map the current yield gap with much focus on improving land management practices. In concert, plant breeding is shifting from creating “specialist” cultivars that require optimal conditions for their performance toward creating “robust” cultivars that can perform optimal in a broad range of suboptimal conditions, with the ultimate goal of closing the yield gap.

4Crop yield is driven by the combination of climate, soil, management, and genetics. Under optimal circumstances the soil provides plants with stability, water, and nutrients. However, soils are heterogeneous environments, strongly influenced by outside factors. Nutrient deficiency, drought, salinity, flooding, and temperature are major drivers of the current and future yield gap. Researchers and breeders work together to develop crops that are able to withstand these stresses (as reviewed in ). However, current crop selection is mainly focused on the shoot, whereas most major drivers of the yield gap affect soil properties, directly influencing the root system. This paper will therefore focus on the potential of optimizing root systems for improving crop abiotic stress tolerance.

5Breeding efforts to improve crop yield are in general focused on aboveground, shoot-related phenotypes, whereas the roots as ‘hidden half’ of the plant are still an under-utilized source of crop improvement. Trials aimed to select for new cultivars with improved crop yield are in general performed under optimal nutrient concentrations, which has often led to selection for smaller and less plastic roots. Moreover, modern cultivars develop in general faster and the earlier initiation of shoot sinks stimulates the investment of biomass into the shoots rather than into the roots. Modern wheat cultivars indeed have smaller root sizes and root:shoot ratios than older ones. Given the crucial role roots play in the establishment and performance of plants, researchers have started ‘the second green revolution’ to explore the possibility of yield improvements through optimization of root systems.

6Because water and nutrients are not evenly distributed in the soil, the spatial arrangement of the root system is crucial for optimal use of the available resources. This spatial arrangement of the root and its components is referred to as root system architecture (RSA). Length, number, positioning, and angle of root components (as described in Figure 1) together determine RSA (Figure 2). These traits determine the soil volume that is explored. In addition, the root surface area depends on root hair development and root diameter. The ability to adjust RSA is an important aspect of plant performance and its plasticity to a large variety of abiotic conditions. Root development is guided by environmental information that is integrated into decisions regarding how fast and in which direction to grow, and where and when to develop new lateral roots. The limits of root system plasticity are determined by intrinsic pathways governed by genetic components. Understanding the development and architecture of roots, as well its plasticity, holds thus great potential for stabilizing the productivity under suboptimal conditions in the root environment. Although, plants are capable of adjusting a wide range of developmental and molecular processes in the root to cope with abiotic stress, this review will mainly focus on the plasticity of RSA, their proposed adaptive values, and its use in the selection and breeding of more robust crops.

7Plants use macronutrients as the basis of proteins and nucleic acids. Especially the availability of phosphorus (P) and nitrogen (N) determine plant performance. Other nutrients are used as co-factors for enzymes or to drive membrane transport. Complications in nutrient acquisition can arise because of nutrient shortage in the soil, but other factors such as pH, the balance of different nutrients and soil composition also play a role. For examples, high salinity can decrease the solubility and thus availability of phosphate.

8Nutrient deficiencies are responsible for the major part of currently observed yield gaps worldwide. estimated that for 73% of the areas with a yield gap bigger than 25%, solely improving nutrient balances in the soil could close this gap. This illustrates the impact of nutrient imbalances and deficiencies on plant productivity. If we also consider the high use of fertilizer in agriculture, improving plants’ capability of dealing with nutrient deficiencies and increasing their of nutrient acquisition is of major importance.

9Nutrients are distributed heterogeneously and often have a strong vertical distribution pattern. Leaching on the one hand and plant cycling on the other hand influence the nutrient distribution pattern. Leaching is caused by vertical water flow and takes nutrients down to lower soil layers, were water flow decreases and nutrients accumulate. Plant cycling is based on nutrients taken up from and cycled back to the soil, which causes nutrients to deplete in the root zone and accumulate in the topsoil. Horizontal distribution of nutrients is mainly dependent on the plant distribution aboveground, leading to higher nutrient accumulation underneath canopies. Vertical distribution depends on the balance between leaching and plant cycling, which differs strongly between nutrients. Low mobile nutrients with a prominent role in plant growth, such as phosphate and potassium, undergo high plant cycling, leading to topsoil accumulation. In contrast, mobile nutrients, such as nitrate and chloride, are subject to leaching leading to accumulation in deeper soils. The challenge for plants is to cope with this heterogeneous and sometimes contrasting distribution of nutrients and other resources. In agriculture, plant cycling is often reduced, due to harvesting of plant material, increasing leaching and loss of nutrients. To cope with this heterogeneity, plants can adapt their RSA to specifically forage those parts of the soils where nutrient availability is high.

10Recently, RSA changes upon a wide range of nutrient deficiencies have been mapped in Arabidopsis growing on agar plates. Each deficiency led to a distinct response in RSA development, which is consistent with the fact that not all nutrients have the same accumulation pattern and thus ask for a different response. For example, the readily available forms of the two most limiting nutrients, nitrate (NO3-) and phosphate (PO43-), have an almost opposite accumulation pattern in the soil. Whereas immobile phosphate accumulates in the topsoil, mobile nitrate quickly leaches to deeper soils. This challenges the plant to respond differently to a deficiency of these nutrients. Fortunately, the RSA responses to these deficiencies have been mapped extensively in both Arabidopsis and crop species, offering us many insights in functional RSA development.

11Phosphate is a building block of, for example, nucleic acids and membrane phospholipids. Because of the high phosphate demand of plants, limitation in phosphate has a strong effect on plant growth (as reviewed in; ). Efficient uptake of phosphate is therefore essential. High plant cycling, in combination with low mobility, leads to accumulation of phosphate in the topsoil. To optimally forage the soil for phosphate, plants need to develop a shallow root system (as reviewed in ). The RSA response to phosphate deficiency in Arabidopsis is well-characterized (as reviewed by ). A strong shift from main root growth to lateral root growth is observed, which leads to a short root with a high number of long laterals (Figure 3A;;;; ). In addition, a strong proliferation of root hairs is observed. These changes result in a shallow root system, optimal for topsoil foraging.

12For maize, a series of papers was published in which the value of certain root traits for phosphate acquisition was evaluated using a set of RILS distinctly different in these root traits. Shallow rooting maize varieties showed increased net phosphate acquisition, corrected for possible higher phosphate investments. A big screen of 242 accessions of maize on high and low phosphate availability confirmed the importance of root plasticity under low phosphate conditions. Yield and biomass was increased for accessions with a higher number of nodal and lateral roots. In addition, dense root hair formation also correlated with higher biomass under low P conditions.

13Shallow root system development is a result of strong investment in lateral root growth. confirmed that in maize enhanced lateral root formation is beneficial for net phosphate acquisition. In comparison to the primary root and other components of the root system, lateral roots are cheap in terms of phosphate use. Similar results were found for enhanced seminal root growth, which is especially important for phosphate acquisition during early seedling development. Several studies show that strigolactones are key regulators of both root and shoot responses to the level of available phosphate. The effect of strigolactones on RSA depends on phosphate availability. Whereas strigolactones inhibit lateral root emergence and elongation and promote primary root elongation when phosphate is sufficient, the opposite is observed when phosphate is depleted. Interestingly, a similar phosphate dependent effect of ABA on lateral root development has recently been observed. The contrasting effect of strigolactones is a result of modulation of auxin distribution and sensitivity, both underlying the strong shift from primary to lateral root growth. Addition of the synthetic auxin NAA doubled expression levels of genes involved in the cell cycle specifically during phosphate starvation. Increased auxin sensitivity during phosphate starvation appears to be explained by increased expression of the auxin receptor TRANSPORT INHIBITOR RESPONSE1 (TIR1), leading to increased degradation of AUX/IAA and released repression on auxin response modules. Interestingly, strigolactones have been shown to be responsible for the increase of TIR1 expression during phosphate limitation.

14The inhibition of primary root growth in Arabidopsis (Col-0) in response to phosphate starvation has been shown to be strong and irreversible. During phosphate starvation, primary root development changes drastically, shifting from indeterminate growth to determinate growth. Preceding this drastic shift, changes in the quiescence center are observed, suggesting an important role during phosphate starvation. Consistently, show that specifically exposing the root cap to low phosphate is sufficient to induce growth arrest in the primary root. Mutants lacking determinate growth in low phosphate conditions show reduced activation of the phosphate starvation rescue system. These findings suggest an important role for the root cap in sensing environmental conditions.

15During phosphate limitation, Arabidopsis develops a high number of long root hairs. Compared to mutants lacking root hairs, wild type plants have a higher phosphate uptake resulting in more plant growth. measured phosphate uptake of root hairs by providing the radioisotope 32P to root hairs of rye plants in the soil. The root hairs contributed to a substantial amount of 63% of total phosphate uptake. Consistently, a mutant of barley lacking root hairs took up half the amount of phosphate compared to the wild type. Under low phosphate conditions, cultivars of barley with long root hairs are able to sustain high yields, whereas cultivars with short root hairs produce substantially less yield. Interestingly, no disadvantage of root hair development under high phosphate availability is found for either Arabidopsis and Barley. As for other root traits, strigolactones seem to play a major role in the regulation of the number and length of root hairs.

16Next to length and number of root components, the angle of the roots also determines whether a root system develops shallow or deep. Roots are able to sense gravity, allowing the main root to grow down into the soil, a response called gravitropism. Although lateral roots are also gravitropic, they typically show a gravitropic setpoint angle (GSA;; ), resulting in non-vertical emergence from the main root (see also salinity and drought sections). Under low phosphate conditions, gravitropism could be expected to counteract development of a shallow root system ideal for topsoil foraging. In accordance, in common bean, development of a shallow root system depends on the ability to adjust the gravitropic offset angle. This ability indeed correlated with its ability to cope with low phosphate conditions. Subsequent investigation of RILs with contrasting root gravitropic offset angles showed a strong correlation with phosphate acquisition and plant growth.

17In contrast to phosphate, nitrate is highly mobile in soils and is therefore prone to leaching. In environments where nitrate is limiting, deeper soil layers can often offer nitrogen supplies. Consistently, availability of phosphate and nitrate has contrasting effects on RSA. Low nitrate availability in general limits plant growth. However, low nitrate availability does not limit primary and lateral root elongation, enabling the root system to reach deeper layers of the soil (Figure 3B;; ). This shift in investment results in an increase in root:shoot ratio. For maize, a monocot species, reaching greater rooting depth requires the development of a lower number of crown roots. Maize genotypes with lower crown root number showed 45% greater rooting depth, which was accompanied with higher N acquisition. The biggest difference in N acquisition was found in deeper layers, emphasizing the importance of a deep root system for nitrogen acquisition.

18Lateral root density is not affected by homogeneous nitrate limitation. Interestingly, in a heterogeneous environment, a strong increase in lateral root density in nitrate patches is observed in both Arabidopsis and maize. When plants are exposed to nitrate patches, lateral root elongation rates outside the patches were strongly decreased, indicating a shift of investment of resources. Plants are thus able to selectively place their roots to efficiently forage the soil. The mechanism of utilization of heterogeneously distributed nutrients by selective placement of lateral roots in or near nutrient enriched patches is best studied for nitrogen. However, selective root placement for a wide range of nutrients was already illustrated in 1975. A limited part of the root system of barley was exposed to high concentrations of phosphate, nitrate, ammonium, and potassium. For all of these nutrients a strong proliferation of lateral roots in the zone of high availability was observed. Growth of lateral roots in other zones was strongly limited. This emphasizes the importance of investigating this response for other nutrients.

19The nitrate transporter NRT1.1 plays an important role in perceiving nitrate levels in the soil. The nrt1.1 mutant displays no increase in lateral root proliferation in nitrate rich patches, while the RSA response to homogeneous nitrate limitation is not affected in this mutant, indicating that this is not an effect of reduced nitrate uptake. Interestingly, NRT1.1 has the ability to transport auxin and this transport is inhibited by nitrate. showed that in nitrate patches, nitrate inhibits auxin transport by NRT1.1 out of lateral root tips and primordia, leading to auxin accumulation and stimulation of lateral root growth. Outside these patches, nitrate levels are low and NRT1.1 prevents accumulation and thus lateral root growth.

20NRT1.1 has been shown to affect expression of several downstream genes involved in nitrate starvation responses, including NRT2.1. NRT2.1 is a major component of high-affinity nitrate uptake in the root. NRT1.1 and NRT2.1 seem to be responsible for repression of lateral root growth outside nitrate patches, based on their mutant phenotypes. Nitrate starvation can trigger ethylene production, a phytohormone that influences root growth. NRT2.1, also induced by nitrate starvation, seems to stimulate ethylene production. Conversely, ethylene inhibits NRT1.1 and NRT2.1 expression, possibly providing a negative feedback loop important for fine tuning responses.

21Besides nutrient limitation, water limitation is the biggest driver of the yield gap. have shown that in 16% of the areas with a current yield gap bigger then 25%, improving irrigation can solely close the gap. In addition, for all investigated areas improving irrigation would decrease the gap. This illustrates the importance of water availability for plants. Plants need water for transport, structure and photosynthesis among other processes. Most crops have high water requirements and are poorly drought resistant. However, irrigation is already responsible for 70% of the total use of available freshwater. The present focus of plant breeders therefore is on improving water use efficiency of crops.

22When water availability is limited, the soil osmotic potential decreases and plants are confronted with osmotic stress. Plants cannot take up water and sometimes even loose water to the soil. The high surrounding osmotic potential leads to loss of turgor, starting in the root. The combination of rapid sensing and signaling, followed by adjustments on both cellular and organ level, can enable the plant to limit water loss and survive drought stress (as reviewed in ). Drought stress leads to distinct changes in RSA, both on whole-root system and sub-organ level.

23Water is generally stored in deeper soil layers, because the topsoil dries more quickly. Plants that develop deeper root systems will have access to water stored in these deeper layers. Among other traits, deeper rooting has been shown to be beneficial for plant production and survival under water limiting conditions (as reviewed in ). For example, the generally deeper rooting mutant extremely drought tolerant1 (edt1) in Arabidopsis shows high drought tolerance. This is explained by the ectopic overexpression of the HD-ZIP transcription factor HDG11, which directly promotes the transcription of genes encoding cell wall loosening proteins. These proteins promote cell elongation in the root, leading to an extended root system. Interestingly, expression of HDG11 in other species such as rice, poplar and cotton, also confers drought tolerance.

24Reaching deeper soils requires a shift from investment in lateral roots to investment in axile roots (Figure 3C). Arabidopsis shows a strong inhibition of lateral root emergence and elongation when grown on agar medium containing an osmoticum, such as sorbitol or mannitol, mimicking osmotic stress. Importantly, showed a possible link between inhibition of lateral root growth on agar and drought tolerance in soil. Mutants performing well under drought conditions in soil, showed high sensitivity to ABA leading to strong inhibition of lateral root length on agar media. In comparison, less tolerant mutants showed no inhibition of lateral root length. ABSCISIC ACID INSENSITIVE4 (ABI4), enhanced by ABA during drought stress, can inhibit PIN1 expression, leading to decreased polar auxin transport and decreased lateral root formation. This mode of action of ABA provides a possible mechanistic explanation for the effect of ABA on lateral root formation.

25Polar auxin transport by influx and efflux carriers determines auxin distribution in the root, which is not only important for LR formation, but also for bending of plant organs by differentially affecting cell elongation. This bending is essential for gravitropism of the main root. Positive gravitropism, growing in the direction of gravity, orientates the root downward and enables penetration of the soil. However, other root components, such as lateral, seminal and crown roots can display very different growth angles, partly suppressing gravitropism. The angle of these roots strongly determines whether RSA develops shallow or deep. In lateral roots PINs determine auxin distribution and thus the GSA. The magnitude of the difference in auxin concentration between the upper and lower side of the lateral root determines how strong a lateral root will bend. As previously described, auxin transport is inhibited during drought stress due to the inhibition of PIN1 expression, which might facilitate increased downward bending of the roots.

26In several crop species increased downward bending of the roots is correlated with drought tolerance. In rice, a strong correlation between the angle of roots and drought tolerance is observed. High expression of the DEEPER ROOTING1 (DRO1) gene in rice, responsible for increased downward bending of the roots by altering the auxin distribution, results in maintained high yield under drought stress. This example indicates that adapting RSA, in this case both using genetic and transgenic approaches, can result in increased drought tolerance. Similar to rice, the angle of seminal roots in wheat cultivars also correlates with drought tolerance. Drought tolerant wheat cultivars develop seminal roots with a narrow angle, growing deeper into the soil.

27Although a strong vertical distribution pattern of water exists, soil heterogeneity in water content exists and sensing of available water is crucial for optimal water uptake. It has been shown that plants are able to partially repress gravitropism and grow toward water, the so-called hydrotropism response (as reviewed in;; ). To investigate hydrotropism in Arabidopsis, different growth systems have been used, in which either salt solutions or agar with sorbitol created a gradient in osmotic potential and thus a gradient in water availability. Arabidopsis was able to redirect growth of its main root away from a low osmotic potential and thus low water availability. This moisture-driven hydrotropic response has also been observed in other species including maize, cucumber, and pea.

28As described previously, the distribution of auxin, driven by polar auxin transport, has a central role in regulating bending of plant organs and response to gravity. Interestingly, hydrotropism seems to be independent from polar auxin transport, as the repression of influx and efflux carriers of auxin do not inhibit the response. Recently, auxin distribution during hydrotropism was measured with the DII-VENUS SENSOR. Indeed, during the first 2 h of hydrotropic response, no change in auxin distribution was observed. In the presence of NPA, an inhibitor of auxin transport, hydrotropic bending was not inhibited. The involvement of auxin through changes in auxin sensitivity or biosynthesis remains ellusive due to contrasting results showing either positive, negative or no effects of inhibition of auxin responses or sensitivity.

29It has been shown that Arabidopsis roots can distinguish a wet from a dry surface and selectively favor development of roots in these wet places over development in dry places. These wet surfaces determine where new lateral root founder cells are formed. have shown that under dry conditions lateral root primordia develop at similar rates as under control conditions. These primordia can subsequently be rapidly induced in zones with high water availability. The combination of formation and emergence of primordia leads to specific root proliferation at sites of high water availability, so-called hydropatterning. This process seems to be independent of the major drought stress hormone, ABA. Further research on this new topic is required to provide more knowledge on how plant roots sense moisture and adjust RSA accordingly.

30Salinity is a major and increasing problem for agriculture. Most crop species are salt sensitive and grow poorly on salinized soils. In 1992, the extent of salinity-affected soils was estimated at 410 billion ha. Although an adequate mapping of the current extent of salinized soils is lacking, over 100 countries are confronted with soil salinization. On a yearly basis between 0.3 and 1.5 million ha of arable land are lost to salinization and another 20–40 million ha are strongly affected by salinity. Although some of these are naturally occurring saline soils, current observed salinization is often the result of irrigation practices. Irrigation in arid zones, accounting for approximately 40% of irrigation worldwide, mobilizes salts stored in the deeper soil layers. In addition, due to freshwater scarcity, an increased use of brackish irrigation increases salt levels even further. The increasing losses of arable land due to salinization ask for the development of salt tolerant crops.

31Similar to drought, salinity can cause problems due to the high osmotic potential in the soil, leading to osmotic stress. In addition, salinity affects plant growth due to the toxicity of high sodium Na+ levels. Na+ toxicity especially causes problems in the shoot by inhibiting photosynthesis among other processes. Na+ is chemically similar to K+ and can interfere with processes in which K+ plays an essential role. The capacity to maintain a low Na+/K+ balance in the shoot has been shown to be closely linked to salt tolerance. Preventing Na+ transport to the shoot is thus very important. The root system is responsible for water uptake, accompanied by dissolved ions including Na+, and thus plays an essential role in preventing Na+ from entering the vascular system and reaching the shoot.

32Salt has a distinct effect on root growth (as reviewed in ). Although, low salt concentrations up to 50 mM can promote plant growth in Arabidopsis, higher salt concentrations have severe negative effects. Both primary and lateral root growth is inhibited during salt stress (Figure 3D; ). In addition, lateral root number specifically decreases in the root zone developed after exposure to salt stress (Figure 3D; ). Most studies show no effect of salt stress on lateral root density, indicating that the decrease in number of lateral roots is related to the inhibition of primary root growth.

33Within seconds after exposure to salt stress, plant signaling is activated. This early signaling leads to adjustments in plant growth (as reviewed in ), starting with a quiescence of growth in all plant organs. The quiescence phase is caused by a temporary inhibition of mitotic activity, leading to lower cell division rates. After the quiescence phase, growth recovers again. However, growth rates only recover to a certain extent, because the inhibition of the cell cycle during the quiescence phase results in fewer cells in the meristem. In addition, mature cell length is smaller in salt stressed roots.

34Quiescence is induced by abscisic acid (ABA), which is rapidly up-regulated under salt stress due to the decrease in osmotic potential. ABA in general inhibits both gibberellin (GA) and brassinosteroid (BR) signaling and stress-induced reduction of growth has been shown to benefit the plant. It is thus proposed that the quiescence phase is essential to induce changes to cope with salt stress. The quiescence phase is followed by a partial growth recovery, that is mainly guided by an increase in GA and BR levels.

35The length of the quiescence phase differs strongly between root components. Whereas quiescence in the main root takes approximately 8 h, this phase can take up to 2 days in lateral roots. In a similar way, the recovery extent of different organs differs. Although overall an inhibition of root growth is observed, there is a distinct difference between the effects of salt on primary in comparison to lateral root growth. have shown that in Col-0 the relative growth rate of the primary root was more strongly affected than the growth rate of the lateral roots. This indicates that the RSA is remodeled during salt stress. The adaptive value of this remodeling with respect to salinity tolerance is still unclear and requires further research.

36In a screen of 32 Arabidopsis accessions, a first indication for a relation between remodeling of RSA during salt stress and salt tolerance was found. The screen revealed four distinct growth strategies during salt stress, depending on the relative inhibition of the number of lateral roots, main root and lateral root growth rates. One of these strategies was correlated with a much lower Na+/K+ level in the shoot, indicating less Na+ uptake and thus a higher tolerance. This strategy is characterized by a strong inhibition of lateral root growth rates, while main root growth rates and number of lateral roots are much less affected (Figure 3D).

37Besides remodeling of the root system during salt stress, plants also show reduced gravitropism under saline conditions. showed that plants can specifically redirect growth away from higher salt concentrations, a response called halotropism. This response was observed in Arabidopsis, tomato and sorghum seedlings, both on agar media and in soil. Similar to gravitropism, auxin redistribution is central in regulating halotropism. Endocytosis of PIN2, an auxin efflux carrier, at the side of high salt concentrations, redistributes auxin in the root. The redistribution of auxin is supported by auxin-induced expression of AUX1, an auxin influx carrier. Both mathematical modeling and experimental data have shown that these processes, together with a transient PIN1 increase, are responsible for the root bending away from salt.

38Part of the salinity response is also triggered by osmotic stress and shows overlap with drought responses. However, the changes in RSA show distinct differences. For example, main root growth is strongly promoted during drought, whereas it is inhibited during salt stress. It is not well-known whether the above described quiescence phase is also displayed during drought stress. Because the osmotic component of salinity is believed to underlie this response, it is worth investigating. For halotropism and hydrotropism, although similar responses, the underlying mechanisms seem to differ. In contrast to halotropism, hydrotropism has shown to be independent of auxin transport. Halotropism is dependent on auxin distribution and occurs only in response to Na+ ions, indicating it is a specific response to high salinity. For drought stress, the function of changes in RSA has been studied extensively, whereas salinity research has been more focused on the underlying mechanistic principles. In future research, studying the overlaps and differences between these stresses can benefit knowledge in both areas.

39Most crop species are highly sensitive to salinity. Tomato serves as a model crop that is widely used to study how salt tolerance can be enhanced in crop species. For a wide range of vegetables, including tomato, grafting is a very effective way to increase crop resistance to biotic and abiotic stresses, without affecting above ground characteristics (see also challenge 3 in section on crop selection). For several salt sensitive commercial tomato cultivars, grafting onto rootstocks of more tolerant cultivars has positive effects on productivity when exposed to high salinity. The Na+/K+ levels in the shoot (scions) indicated that the tolerant rootstocks prevented Na+ reaching the shoot, illustrating the importance of the root system for salt tolerance. Unfortunately, only little is known about RSA development of crops during salt stress. In rice, rye, and maize inhibition of root length has been observed under high salinity. Similar to Arabidopsis, maize shows a quiescence phase in response to exposure to high salinity, followed by recovery. In rye, the reduction in root growth is related to a reduction in cell division and an increase in cell death. Further research on remodeling of the root system of crop species will be necessary to use our current knowledge in Arabidopsis to improve crop tolerance to salinity.

40Already 10% of cultivated land surface is so poorly drained that waterlogging, leading to anoxic conditions in the root zone, causes crop yield losses. Twenty percent of agricultural land in Eastern Europe and the Russian Federation and 16% in the USA are too wet for optimal plant functioning. As climate change is expected to lead to more frequent heavy precipitation during the plant growth season in some areas, these problems will increase. Flooding and hypoxia impose an immediate and dramatic limitation for root functioning. Limiting the oxygen supply to root cells causes an almost instantaneous arrest of root growth (as reviewed in ). Switching from aerobic respiration to the glycolytic generation of ATP leads to a severe reduction in energy available for maintenance, growth and ion uptake. Of these three different functions, growth takes 20–45% of ATP generated through respiration. Balancing the demand for energy with the reduced production through glycolysis could therefore also cause limiting root growth. Arrest of root growth could, however, also be caused by accumulation of products of anaerobic metabolism. A lethal drop in pH of the cytoplasm can occur when protons accumulate in the cytoplasm and the vacuole. In Phragmites australis addition of low molecular weight monocarboxylic acids, such as acetic acid, propionic acid, butyric acid and caproic acid, and sulfide, at concentration levels that have been measured in situ, arrested root elongation. As the rate of root elongation is one of the most important parameters determining nutrient uptake rate, flooding-induced inhibition of root growth ultimately would lead to nutrient limitation and negatively impact the survival of the whole plant.

41One of the best-studied adaptations of plants to flooding conditions is the formation of aerenchymatic tissue in the root, which provides an alternative pathway for the supply of oxygen to the root tissue. This requires that new, well-adapted, adventitious roots are being formed. In these roots, axial oxygen loss can be kept to a minimum so that the root tip becomes a well-oxygenated micro-climate. Most of the disadvantages for root metabolism imposed by the flooding-induced hypoxic conditions are thereby ameliorated. In monocot plants the formation of new nodal roots, replacing the old seminal roots and often containing aerenchyma, can be stimulated, leading to superficial rooting patterns. If plants are not capable of increasing their oxygen supply through aerenchymous conducts in the root or by placing new roots close to the soil surface where the oxygen level might be higher, survival of flooding is unlikely.

42Temperature is a key abiotic factor involved in seed germination and subsequent root system development during early seedling establishment. The temperature of the soil fluctuates by sinusoidal oscillations on a diurnal scale. However, depending on soil depth, changes in soil temperature are delayed and much lower in amplitude than variations in the atmospheric temperature. The root-zone temperature (RZT) thus fluctuates daily, seasonally, and with soil depth. Depending on the season and the time of the day, the temperature of the root environment can be significantly different than the atmospheric temperature experienced by the shoots. The RZT directly affects root development, uptake and upward transport of water and nutrients, phytohormone production, which in turn affect water status, stomatal conductance, photosynthesis, biomass partitioning, leaf, and shoot growth. Plant species clearly differ in their optimal temperature range for root development; e.g., oat 4–7°C, wheat 14–18°C, pea 15–20°C, tomato 22–25°C, sunflower 25–30°C, and cotton 32–35°C. Root:shoot ratios usually increase under unfavorable RZTs as long as temperature limits for root development are not reached. This adaptation in root:shoot ratio may overcome restrictions in water and nutrient uptake due to increased water viscosity and/or decreased root hydraulic conductance. Global climate change is likely to exacerbate plant abiotic stress in coming decades by increasing fluctuations in soil temperature and (related) water availability. Breeding crops with a broader root-zone temperature optimum is therefore of significant importance to improve future plant performance. Improved knowledge of the key regulators for RSA optimization would support these breeding efforts.

43The exposure of both mono- and dicot plant roots to temperatures below or above their optimum temperature generally decreases (i) primary root length, (ii) lateral root density (numbers of lateral roots per unit primary root length) and (iii) the angle under which lateral roots emerge from the primary root, whereas the average lateral root length is unaffected. In addition, roots suffering from supraoptimal temperature stress start to initiate second and third order laterals and are characterized by an increased average root diameter. In general, the modulating effect of sub- and supraoptimal RZTs on RSA development reduces the volume that roots may access for the uptake of water and nutrients. However, root temperature was kept spatially uniform in all these studies. Remarkably, monocot barley plants exposed to a vertical RZT gradient of 20–10°C showed increased shoot and root dry masses of 144 and 297%, respectively, and a 161% increase in root:shoot ratio compared with plants grown at a uniform RZT of 20°C. Barley exposed to the vertical RZT revealed also accelerated tiller formation. The higher root biomass of plants grown at the vertical RZT gradient was not the result of longer roots but was associated with a higher proportion of thicker roots. Additionally, root systems developed under a vertical RZT gradient were much stronger concentrated in the upper 10 cm of the soil substrate gradient and their N and C concentrations were significantly lower than under uniform RZT conditions. These data clearly demonstrate that knowledge gained from experiments with uniform RZTs cannot simply be extrapolated to the field where roots experience vertical temperature gradients.

44The temperature dependence of RSA development shows strong inter- and intraspecific variation. The temperature plasticity of the RSA is most extensively studied in maize. In this monocot species, the total lateral root length correlated significantly with improved photosynthesis-related traits and dry matter accumulation at suboptimal growth temperature. A high density of long lateral roots was therefore regarded as a promising trait to improve early seedling vigor at suboptimal soil temperatures. Nevertheless, breeding has to focus on optimizing RSA over a broad range of RZTs as roots also experience temperatures in the optimal- or even supraoptimal range during the entire growth season. At high (root-zone) temperatures the development of long axile roots is of greater importance than lateral roots to facilitate appropriate water uptake from the lower soil layers in times of drought stress. A schematic overview of general observed effects of non-optimal temperatures on RSA and its adaptations to broaden the RZT range for optimal plant performance are presented in Figure 4.

45To optimize RSA over a broader temperature range, provided the prove-of-concept that hybrids of southern dent and northern flint maize inbred lines, which contrast in temperature dependence of axile and lateral root elongation rates, showed improved rooting potential across the sum of all temperatures. Application of this heterosis effect can lead to hybrids that can perform well in a broader range of temperature conditions, thereby improving the robustness of whole-plant performance.

46The primary stunting effect of sub- and supraoptimal temperatures on RSA is caused by inhibition of root elongation. In Arabidopsis accessions, the relative decrease in root elongation rates after transfer from 21 to 10°C were not significantly correlated with the average temperature during the growing season of the specific ecotype, suggesting that primary root growth at 10°C is not a key factor in adaptation to colder habitats. Within tomato, however, the relative inhibition of root elongation and root growth rates at low temperatures were indicative for the difference in chilling tolerance between domestic cultivars and high-altitude accessions of the wild tomato Solanum habrochaites. Dynamic changes in temperature severely affect the elongation rate of root cells rather than the length of the elongation zone. In the short-term (hours), inhibition in root cell elongation by low temperature is related to a decrease in the in vivo extensibility of the cell wall. Gravitropism experiments with Arabidopsis roots demonstrated that acute cold stress (4°C) selectively inhibits the basipetal auxin transport due to blocking the intracellular trafficking of a subset of proteins that include auxin efflux carriers (PIN2 and PIN3). As a consequence, auxin accumulates to a level at which root cell elongation is inhibited. When plant roots have enough time to acclimate to a constant low RZT (weeks), cell elongation rates increase again and the length of the elongation zone expands. This may explain the strong linear relationship between temperature and elongation rates of both primary and later roots directly after germination and its disappearance later on during seedling establishment.

47Variation in root elongation rates among Arabidopsis accessions correlated at optimal temperature with the production rate of cells within the root meristem. Cell production, in turn, was determined by variation in cell cycle duration and, to a lesser extent, by differences in the number of dividing cells. Cell production rates strongly correlated with the activity of the cyclin-dependent kinase (CDKA). Low temperature decreased the division potential of the root meristem in Arabidopsis by reducing both the meristem size and cell number. The repression of the division potential of root meristematic cells at a suboptimal temperature of 16°C could be ascribed to a reduced accumulation of auxin in the root apex. Long-term (7 days) exposure to 16°C inhibited the expression of PIN1/3/7 and auxin biosynthesis-related genes suggesting that auxin transport and biosynthesis both contribute to the low-temperature mediated reduction of auxin accumulation in roots tips. Root length and meristem cell number of ARABIDOPSIS RESPONSE REGULATOR 1 (arr1-3) and 12 (arr12-1) cytokinin signaling mutants were much less susceptible to low temperature than wild-type roots. This difference was related to higher PIN1/3 expression in the mutants, which in turn resulted in a less pronounced reduction in auxin accumulation. These data, together with the results obtained with the cytokinin signaling mutant ahp1-1 ahp2-1 ahp3, strongly suggest the involvement of cytokinin signaling in the modulation of RSA development at low temperature.

48High RZT (40°C) reduced the elongation and cell production rate of Sorghum seminal roots with 14 and 26%, respectively, for every 2 days of exposure. In contrast to low temperatures, the underlying inhibitory effects of high temperatures and heat stress on root elongation are poorly studied. The limited information that is available in the literature excludes the involvement of altered IAA transport or levels, but supports the involvement of increased ethylene levels. Inhibitors of ethylene biosynthesis partly alleviated the effect of high RZT on root elongation, stomatal conductance and shoot water status, however, they failed in ameliorating the negative effects on photosynthesis and biomass accumulation. This points to a non-stomatal limitation of photosynthesis mediated by high temperature-induced changes in nutrient uptake.

49This review presents a number of examples in which plasticity of RSA traits considerably impact a plant’s capability to cope with one or more abiotic stresses. These examples emphasize the great potential that selection on RSA traits holds for crop improvement. However, the aboveground focus of crop selection is not without reason. In this concluding section we will discuss three major challenges breeders face when applying selection for RSA in their crop improvement programs and possible ways to tackle these.

50The most prominent challenge for crop selection on RSA is uncovering the hidden world of plant roots. Whether crops are grown in fields or in greenhouses, roots are usually grown in a substrate, which prevents easily screening their properties. The growth substrate also greatly affects how roots develop. The currently most common method to investigate RSA, growth on agar medium, is very artificial. Most often roots grow in light, with an excess of sucrose, in 2D and the humidity inside the Petri dish is almost saturated. Effort is taken to improve this system, for example by shielding the roots from light. Although agar media provide an easy, adequate and cheap method that can be used for research on Arabidopsis in the lab, its use in crop selection is not straightforward.

51In the last decade a wide range of new and improved methods to research roots in a more natural environment have been developed (as reviewed in;;; ). Most systems are based on either a transparent growth medium or a medium from which the roots can easily be removed without damage. Agar and other gel-like mediums are suited for imaging during growth, although the resistance of the medium influences root growth and the humidity in these substances is very high. A good alternative is hydroponics, in which the root is growing inside a nutrient solution. Hydroponics is also used in greenhouse culture, making it highly relevant for crop selection. This system also eases harvesting roots for different purposes and measuring exudates of roots. However, roots develop very differently, because resistance is lacking and humidity and nutrients are dispersed homogeneously. In addition, a good supply of oxygen is essential to prevent oxidative stress. A third alternative which is also very promising for automated imaging is aeroponics. In this system roots are grown in water-saturated air created by for example spraying with water and nutrients. This system lacks any material to grow in, which eases imaging. However, without much resistance, roots grow very vast and can have problems extending their root system to the sides against gravity. Last, root systems can be grown inside soil, which is of course most realistic for field crops and many greenhouse crops. However, non-destructive imaging inside soil asks for imaging methods that reach further than a simple camera. Several groups have recently reported the use of X-ray and MRI scans to image roots inside the soil. Although these methods are more expensive, they offer great opportunities for automated imaging. An alternative method is GLO-Roots, based on luminescence genes expressed inside roots. This system visualizes the root system through a thin layer of soil. For fundamental research labs, this is more feasible and also offers the opportunities to image the expression of certain genes in the root system. For root breeding, this is less interesting, because the plants are genetically modified and grown in 2D systems.

52As more methods come available to study the root system and also methods are developed suitable for high-throughput screening of root systems, the need for good root image analysis software is growing. A wide range of root image analysis software exists (as reviewed in;; ). These tools range from automated to non-automated. For a limited amount of data, non-automated software prevents mistakes and gives the user a lot of freedom. However, the analysis is very time consuming and is therefore not suited for large datasets. Automated software can analyze a large dataset rapidly, but especially in complex root systems the analysis is limited to global data such as rooting depth and width. In semi-automated software, such as SmartRoot and EZ-rhizo, the level of user interactions is greater to ensure a lesser degree of analysis errors. Again, this will be more time consuming for larger root systems. In addition, when observing very large root systems, it is even hard to separate roots by eye. Therefore, the development of improved methods of root image analysis has high priority for the field.

53Above described methods are all suited for 2D images of root systems. When simplifying root growth to a 2D system, spatial orientation of roots gets lost. Therefore, new methods such as growing roots in gel cylinders and using X-ray to image through soil will offer sophisticated opportunities to grow and image roots in 3D (as reviewed in ). Although only limited options for reconstructing and analyzing 3D images are currently available, it might eventually be easier to analyze 3D than 2D images, because overlapping and clumping together of roots will be much less common. Developing a good automated imaging analysis set-up of root systems can offer great advancements in crop selection and would be an entirely feasible investment for breeding companies.

54Although the complexity of the combination of different biotic and abiotic stresses is not restricted to the root system, it does make selecting on RSA more challenging. The described RSA responses are mostly known for single stresses and some of the responses are very contrasting. A good example for this is that in drought-tolerant cultivars with a deep-rooting water-conserving phenotype, less root mass is available to forage for phosphorous at shallow depths. Certain stresses tend to occur together often and therefore it might be useful to further investigate the specific RSA response to these conditions. A good example is salt stress and phosphate starvation, as phosphate ions tend to precipitate in saline soils and become unavailable to plants. Both stresses have contrasting effects on several RSA traits and the inhibiting effect of salt on lateral root development might even further limit phosphate uptake. Crucial in crop breeding aimed to optimize RSA is the availability of variation and plasticity in RSA, as observed among Arabidopsis accessions, related tomato species and wheat varieties. Recently, studied the natural variation in the response of 330 Arabidopsis accessions to the combination of salinity and phosphate starvation. In general, responses to salt stress were favored and especially lateral root growth was strongly inhibited. However, not all accessions showed the same response and this natural variation was associated with 13 genetic candidate loci for integrating the plants’ response to combined stress. For many crops, however, the natural variation in RSA is currently still underexploited. Moreover, we need to advance our understanding of the adaptive value of genetically determined differences in RSA on the level of crop performance, marketable yield and fruit quality in targeted root environments and growth conditions.

55Because the complexity of experiments and screenings increases with every additional variable, modeling can provide very useful tools to support research and breeding. A wide range of plant models on different scales is available to the community. These models should now be integrated with a multiscale modeling approach in which developmental processes, RSA, outside environmental factors and plant performance are connected. Current models, however, are often not easy to integrate. When developing a model, the general challenge is to make it comprehensive, widely applicable and simple. For models describing RSA, most are falling short in one of these requirements. Some are only applicable for a certain species or stage of life, which limits the use for crop systems. As soon as models tend to be more widely applicable or incorporate more conditions, they tend to become more complex and the number of parameters increases. This decreases the ease of interpretation and especially the ease of integration into a larger model (including soil and plant performance models). The last few years, a range of more simple models have been published. These models are often based on a few simple rules. For example, ArchiSimple bases root system development on the fact that the growth rate of a root depends on the thickness of the root. By using a simple and widely applicable model, it will be possible to implement models of soil behavior and of plant productivity. Some of the root models have already been integrated with models for changes in the soil (as reviewed in;; ) and show to be very promising in predicting the responses of the root system. One example is the ROOTMAP model, which integrates soil-water-nutrient dynamics with root growth responses in a three dimensional system. Simulations are based on a simple external supply/internal demand principle. The model has shown its use in simulating the efficiency of different RSA types in both heterogeneous phosphate and nitrate supplies. A good example of how such a model can provide valuable information is given by, who show how the model can guide the efficient placement of phosphorus fertilizer. In a similar way, this kind of model could guide in selecting a preferred RSA and potentially even predicting possibly involved processes.

56A model that integrates soil behavior, RSA and plant performance will offer a lot of information to breeders. To confirm whether a root system is advantageous under certain stresses as predicted by the model, RILS with contrasting root systems could be exploited (as illustrated in;; ). If indeed the predicted root system is advantageous, breeders could screen for this type of root system in a high throughput phenotyping system as described in the previous section. This screen can then be used for determining genes that are associated with this trait and can be used as targets for further selection. The model could also predict whether changing certain root system characteristics would negatively influence productivity. Of course, developing such a model is a major challenge still, but investments in developing a good model will be able to speed up crop selection and could model complex combinations of stresses.

57Crop selection on aboveground traits has lead to high-yielding cultivars and crop selection for a certain RSA may come with costs. The root:shoot ratio is known to increase during almost every abiotic stress that has been discussed in this review. On the other hand, selection on RSA does not equal selection for a bigger root system. Our examples show shifts between different root organs, rather than shifts in biomass partitioning between the shoot and the root. In this way, deeper rooting in rice, caused by expression of HDG11, confers drought tolerance without any yield penalty. However, unwanted side effects of selection are not uncommon. An excellent tool to address this problem is to make use of grafting.

58Grafting is the process in which the root system (rootstock) of one plant is connected to the shoot (scion) of another (as reviewed in ). This process naturally occurs in some tree species and this phenomenon may have triggered the development of grafting in Asia where it is now used in agriculture for over 2000 years to improve plant production. In woody perennial crops as well as in annual vegetable crops, the selection and breeding of suitable rootstocks offers a powerful tool to sustain and expand the cultivation under suboptimal growth conditions. Grafting has the advantage that not every cultivar needs RSA optimization separately, allowing improvement of rooting and (a)biotic stress tolerance of already existing elite cultivars. As such, grafting is considered as a surgical and fast alternative to breeding. Designing rootstocks for specific environments is becoming a feasible target to face future cultivation problems all around the world associated with global climate change (drought, salinization, occurrence of temperature extremes; ). Important in this respect is to gain more knowledge of (i) the natural variation in RSA that exists within crops, and (ii) by what communication mechanisms the root(stock) modulates the shoot (scion) phenotype and performance, and visa versa. In this way, grafting can rapidly advance our understanding of the adaptive value of differences in RSA on the level of shoot performance, marketable yield and fruit quality under targeted growth conditions.

59A key aspect for engineering better performing crops via RSA optimization is improved understanding of the regulatory processes and underlying genetic components that regulate root growth. Root growth regulation, and its response to changing environmental conditions, is a highly complicated process that is controlled at many different levels by complex actions of gene networks in both time and space. Advances in this area are merely derived from work in Arabidopsis (as reviewed in; ). It is expected that due to the increasing number of highly efficient root phenotyping platforms, the use of GWAS for root traits, the increasing available functional genomics resources for roots, and the development of smart root model systems, much progress in our understanding of control mechanisms involved in root development will be achieved over the next 5–10 years.

60Although Arabidopsis is often studied under artificial conditions, it is these conditions that make it possible to investigate the partly discussed mechanistic and cellular base behind the observed RSA responses. For crop species only limited information on these processes is available. Interestingly, most plasticity in RSA responses overlaps between our model species and crops, even independent of differences between monocots and dicots. Sparsely investigated functionality of RSA in Arabidopsis supports the results found in crops and conversely sparsely investigated molecular insights in crops confirmed results already established in Arabidopsis. Of course, not all mechanisms, responses and genes can be transferred from Arabidopsis to crops, but taken together the reviewed research, Arabidopsis proves to provide very valuable information for the development of crops able to withstand a wide range of abiotic stresses. This review stresses the importance of incorporating RSA into current crop selection, but we should not forget the wonderful tools we already have. Incorporating RSA into current crop selection also means incorporating Arabidopsis research into the current breeding pipeline, possibly even more then for aboveground traits.

61All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.

62The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.