RootScape: A Landmark-Based System for Rapid Screening of Root Architecture in Arabidopsis

The architecture of plant roots affects essential functions including nutrient and water uptake, soil anchorage, and symbiotic interactions. Root architecture comprises many features that arise from the growth of the primary and lateral roots. These root features are dictated by the genetic background but are also highly responsive to the environment. Thus, root system architecture (RSA) represents an important and complex trait that is highly variable, affected by genotype × environment interactions, and relevant to survival/performance. Quantification of RSA in Arabidopsis (Arabidopsis thaliana) using plate-based tissue culture is a very common and relatively rapid assay, but quantifying RSA represents an experimental bottleneck when it comes to medium- or high-throughput approaches used in mutant or genotype screens. Here, we present RootScape, a landmark-based allometric method for rapid phenotyping of RSA using Arabidopsis as a case study. Using the software AAMToolbox, we created a 20-point landmark model that captures RSA as one integrated trait and used this model to quantify changes in the RSA of Arabidopsis (Columbia) wild-type plants grown under different hormone treatments. Principal component analysis was used to compare RootScape with conventional methods designed to measure root architecture. This analysis showed that RootScape efficiently captured nearly all the variation in root architecture detected by measuring individual root traits and is 5 to 10 times faster than conventional scoring. We validated RootScape by quantifying the plasticity of RSA in several mutant lines affected in hormone signaling. The RootScape analysis recapitulated previous results that described complex phenotypes in the mutants and identified novel gene × environment interactions.Roots have a crucial impact on plant survival because of their major functions: anchorage of the plant in the soil, water and nutrient acquisition, and symbiotic interaction with other organisms (Den Herder et al., 2010). One important characteristic of root systems is the manner in which the primary and lateral roots comprise the superstructure or root architecture. Root architecture is an ideal system for studying developmental plasticity, as it continually integrates intrinsic and environmental responses (Malamy, 2005), which represents a vital and dynamic component of agricultural productivity (Lynch, 1995).Root system architecture (RSA) is defined as the spatial configuration of the roots in their environment (Lynch, 1995). The complexity of RSA was initially appreciated several decades ago, and terms like morphology, topology, distribution, and architecture were often used to describe the nature of RSA (Fitter, 1987; Fitter and Stickland, 1991; Lynch, 1995). These early reports argued that simple traits like root mass are insufficient to describe roots, because they do not capture the spatial configuration of roots in the soil, which is critical to plant performance (Fitter and Stickland, 1991). Root systems are integrated organs that adopt specific architectures to maximal foraging of the heterogeneous soil environment in different ways (Fitter, 1987; Fitter and Stickland, 1991; Lynch, 1995). More recently, new approaches have incorporated the measurement of many individual developmental traits that together comprise RSA (De Smet et al., 2012; Dubrovsky and Forde, 2012). For example, one recent report identified three fundamental components of RSA in generating complex topologies, including the contribution of lateral axes to branching, the rate and path of growth of the axis, and the increase in root surface area (Topp and Benfey, 2012). Thus, RSA is an important and complex trait that requires convenient measurement methods for rapid screening of diverse plant mutants and genotypes.With increasing research in RSA in the genetically tractable model plant Arabidopsis (Arabidopsis thaliana), the need for high-throughput methods of root phenotyping has dramatically increased over the years. Consequently, different methods and approaches have been developed in order to address this demand. Currently, three major approaches for phenotyping RSA are used (for review, see Zhu et al., 2011; De Smet et al., 2012). The first group of methods uses classical measures of RSA, which involve measurements of individual root traits. These methods often use software to manually draw the RSA onto digital two-dimensional images to quantify root length and number (Abramoff et al., 2004; http://www.machinevision.nl). These traditional methods provide the most accurate measurements of the root system but have a major disadvantage in being extremely time consuming.The second group of methods utilizes advanced semiautomated software for RSA measurements like EZ-Rhizo (Armengaud et al., 2009). EZ-Rhizo also uses digital two-dimensional images of plants grown on vertical plates (similar to the classical methods above) but is faster and produces different traits and basic statistics. The method works best when root features do not physically overlap, but we have found root overlap to be common when working with Arabidopsis plants older than 10 d. Other recent programs also provide semiautomated analysis of RSA, including RootReader2D (http://www.plantmineralnutrition.net/rootreader.htm) and SmartRoot (Lobet et al., 2011). However, while completely automated detection is potentially the highest throughput, we found that the root surface detection step is frequently prone to failure when using both of these programs, even after considerable adjustment by the user, where root features are missed or background noise is incorrectly labeled as roots.Finally, in a third group, recent developments include three-dimensional analysis of RSA of plants grown on transparent gel cylinders or in soil. The three-dimensional gel-based imaging approach is reported to be suitable for high-throughput phenotyping (Iyer-Pascuzzi et al., 2010). However, this approach requires special equipment, and imaging the root system of single plants can take 10 min (Iyer-Pascuzzi et al., 2010). X-ray computed tomography (Perret et al., 2007; Tracy et al., 2010) and magnetic resonance imaging (Van As, 2007) also provide highly detailed three-dimensional RSA analysis, but they require long scanning times and are extremely expensive and inaccessible. Most laboratories still utilize relatively convenient, inexpensive, and rapid two-dimensional phenotypic characterization of RSA, at least for initial screening purposes.The aim of this work is to address the need for a simple method to measure many different aspects of root architecture for high-throughput laboratory screening of mutants and genotypes in Arabidopsis. Here, we describe a landmark-based allometric (size and shape) approach called RootScape, a user-friendly software platform that enables rapid, comprehensive, and integrative phenotyping of the RSA in Arabidopsis. Unlike recent methods that collect information on different root traits to describe the RSA, RootScape places user-defined root landmarks on a two-dimensional grid to measure root architecture as a single integrated root system. The method employs rapid manual placement of root system landmarks. This manual step avoids one of the most problematic steps in automated image analysis (recognition of the root surface), providing a simple tool that does not require image processing. This method uses simple, two-dimensional digital images of the root system and a 20-point landmark model created in AAMToolbox, a freely available MATLAB plugin. While in-depth developmental analysis of root systems will often require knowing the contribution of individual traits, RootScape is a rapid method to access the holistic contribution of many individual root traits to RSA and to capture the overall property of the spatial configuration of roots in the soil (Fitter and Stickland, 1991). To demonstrate its utility, we used RootScape to quantify the root plasticity of Arabidopsis plants (Columbia [Col-0]) grown on four different media and compared the RootScape results with conventional measurements of individual root traits captured using the Optimas6 image-analysis software or Image J (Abramoff et al., 2004). This analysis showed that by measuring integrative root traits using RootScape, we could capture the vast majority of the individual trait variation, as verified by multiple regression analysis. Additionally, we tested the ability of RootScape to quantify the plasticity response in Arabidopsis mutants defective in hormone signaling. For this analysis, wild-type Col-0 and three hormone signaling mutants (auxin-resistant4 [axr4], abscisic acid insensitive4 [abi4], and cytokinin response1 [cre1]) were treated with auxin, cytokinin, or abscisic acid (ABA) versus controls. Statistical analyses (ANOVA/multivariate ANOVA [MANOVA]) allowed us to confirm most of the previously known interactions of genotype with these distinct environments and to potentially identify novel ones. Thus, we demonstrate that RootScape can be used as a rapid and efficient approach for quantifying the plasticity of the RSA in mutant (or ecotype) backgrounds of Arabidopsis and can identify new conditional root phenotypes.     

Plasticity of the Arabidopsis Root System under Nutrient Deficiencies

Plant roots show a particularly high variation in their morphological response to different nutrient deficiencies. Although such changes often determine the nutrient efficiency or stress tolerance of plants, it is surprising that a comprehensive and comparative analysis of root morphological responses to different nutrient deficiencies has not yet been conducted. Since one reason for this is an inherent difficulty in obtaining nutrient-deficient conditions in agar culture, we first identified conditions appropriate for producing nutrient-deficient plants on agar plates. Based on a careful selection of agar specifically for each nutrient being considered, we grew Arabidopsis (Arabidopsis thaliana) plants at four levels of deficiency for 12 nutrients and quantified seven root traits. In combination with measurements of biomass and elemental concentrations, we observed that the nutritional status and type of nutrient determined the extent and type of changes in root system architecture (RSA). The independent regulation of individual root traits further pointed to a differential sensitivity of root tissues to nutrient limitations. To capture the variation in RSA under different nutrient supplies, we used principal component analysis and developed a root plasticity chart representing the overall modulations in RSA under a given treatment. This systematic comparison of RSA responses to nutrient deficiencies provides a comprehensive view of the overall changes in root plasticity induced by the deficiency of single nutrients and provides a solid basis for the identification of nutrient-sensitive steps in the root developmental program.Plant survival and performance are highly dependent on the plant’s ability to efficiently explore the soil in the search for water and minerals. Thus, root growth and architecture are extremely relevant for the plant’s adaptation to the growth medium, as they determine the soil volume that a plant is able to explore at a given time. Root system architecture (RSA) represents the spatial arrangement of roots of different ages and orders (Lynch, 1995; Osmont et al., 2007) and is determined by genetic factors and the integration of environmental cues (Malamy, 2005). The genetic component determines the fundamental morphology and blueprint of a plant’s root system, whereas environmental cues shape root architecture by modifying the intrinsic genetic program. The existence of this additional level of regulation allows plants to display a high level of root plasticity, which reflects the shape, three-dimensional distribution, branching pattern, and age of the primary and postembryonically generated roots (Pacheco-Villalobos and Hardtke, 2012). The dynamic modulation of RSA is based on the intrinsic developmental nature of the different components of the root system. In fact, the primary root (PR) is established during embryogenesis, while the lateral roots (LRs) that originate from the PR develop postembryonically (Osmont et al., 2007; Péret et al., 2009). These highly dynamic changes in the overall RSA throughout time finally determine root plasticity and allow plants to efficiently adapt to environmental constraints.Nutrient availability can exert a profound impact on RSA by altering the number, length, angle, and diameter of roots and root hairs (for review, see Forde and Lorenzo, 2001; López-Bucio et al., 2003; Malamy, 2005; Osmont et al., 2007). In fact, plants can respond to the heterogenous availability of resources by allocating roots where the most favorable conditions are found (Zhang and Forde, 1998; Linkohr et al., 2002; Remans et al., 2006; Lima et al., 2010; Giehl et al., 2012). When grown under limited phosphorus (P) availability, roots exhibit a shallower architecture that results from the inhibition of PR elongation and the concomitant increase in LR formation (Williamson et al., 2001; López-Bucio et al., 2002; Sanchez-Calderon et al., 2005). Such an architectural rearrangement of the root is thought to improve the plant’s ability to forage P from the usually P-enriched topsoil horizon (Lynch and Brown, 2001; Rubio et al., 2003; Zhu et al., 2005). In contrast to low P, reduced nitrogen (N) availability stimulates PR and particularly LR elongation but not LR initiation (Linkohr et al., 2002; López-Bucio et al., 2003). However, it is noteworthy that under severe N shortage, LR formation is almost completely absent (Krouk et al., 2010), suggesting that plants require a certain level of N to sustain an active foraging strategy. These examples indicate that the availability of different nutrients can evoke distinct effects on RSA that depend upon which nutrient is supplied and the concentration of the supplied nutrient.Unfortunately, for the majority of the nutrients, a more detailed analysis of the architectural modifications under deficient conditions is still missing. In fact, most studies describe the effect of nutrient deficiencies on root growth and development only in terms of root biomass or total root length (Hermans and Verbruggen, 2005; Hermans et al., 2006; Richard-Molard et al., 2008; Jung et al., 2009; Cailliatte et al., 2010). Thus, important features of the root system are not comprehensible from these rather basic measurements. The characterization of RSA in more detail appears justified due to the positive correlations found between single root characteristics and plant yield, especially when the supply of water or mineral resources was limited (Landi et al., 2002; Tuberosa et al., 2002; Manschadi et al., 2006; Kirkegaard et al., 2007; Steele et al., 2007). Although a large number of studies have been conducted on the root development of grasses (Hochholdinger and Tuberosa, 2009; Iyer-Pascuzzi et al., 2010; Pacheco-Villalobos and Hardtke, 2012), our understanding of the molecular players involved in the regulation of root growth and development has benefited most from studies of the reference plant Arabidopsis (Arabidopsis thaliana) grown under controlled conditions to minimize variability. However, imposing consistent nutrient deficiencies presents an experimental challenge as long as plants are grown on agar medium, which is the method of choice to preserve the spatial arrangement of the root system and access a larger number of root traits.A major drawback of agar and agarose media is their inherent nutrient load, such that traces of nutrient contamination must often be made unavailable to plants, for example by adding chelating agents to lower the free activities of micronutrients (Bell et al., 1991; Yang et al., 1994; Rengel, 1999). Additionally, in many cases, symptoms of deficiency are only observed in mutants impaired in the uptake of the nutrient in question (Tomatsu et al., 2007; Mills et al., 2008; Assunção et al., 2010). In general, gelling agents may contribute considerable amounts of nutrients (Debergh, 1983; Scholten and Pierik, 1998), hampering the occurrence of deficiency for specific nutrients (Jain et al., 2009). Thus, it becomes crucial to select the most suitable gelling agent when particular nutrient deficiencies are to be obtained. This is particularly relevant as strategies depending upon the use of gelling media are being developed to overcome the bottleneck that often limits RSA traits from being characterized in high-throughput phenotyping studies (Iyer-Pascuzzi et al., 2010; Clark et al., 2011).In our approach to compare RSA under different nutrient deficiencies in Arabidopsis plants grown on solid medium, we first identified the most appropriate conditions for producing nutrient-deficient plants on agar plates. Once identified, these conditions allowed us to characterize the effects of 12 deficiencies at four intensity levels on the RSA by measuring seven root traits. These measurements, in combination with biomass and elemental concentrations, allowed us to determine the nutrient-specific effects on particular parameters of the RSA and thus to describe the root plasticity of Arabidopsis and analyze the underlying traits under different nutrient deficiencies.     

Quantitative Trait Locus Mapping Reveals Regions of the Maize Genome Controlling Root System Architecture

The quest to determine the genetic basis of root system architecture (RSA) has been greatly facilitated by recent developments in root phenotyping techniques. Methods that are accurate, high throughput, and control for environmental factors are especially attractive for quantitative trait locus mapping. Here, we describe the adaptation of a nondestructive in vivo gel-based root imaging platform for use in maize (Zea mays). We identify a large number of contrasting RSA traits among 25 founder lines of the maize nested association mapping population and locate 102 quantitative trait loci using the B73 (compact RSA) × Ki3 (exploratory RSA) mapping population. Our results suggest that a phenotypic tradeoff exists between small, compact RSA and large, exploratory RSA.Maize (Zea mays) serves a key role in food, feedstock, and biofuel production throughout the world. To date, maize improvement through breeding has kept pace with the increasing demand for this crop (faostat3.fao.org). This feat has been accomplished through the utilization of the tremendous genetic diversity in maize (Flint-Garcia et al., 2005; Jiao et al., 2012), but increasing environmental pressures and a growing global population will require unprecedented gains in yield in the coming years. In the last decade, researchers have begun to explore the possibility of yield improvements through the manipulation of root systems, for example through breeding for roots better able to cope with drought (Uga et al., 2013) and flooding (Jackson and Armstrong, 1999), the use of plant growth-promoting rhizobacteria (Silby et al., 2009), or increasing nutrient use efficiency (Garnett et al., 2009). The potential of belowground solutions to enhanced plant productivity has driven the development of numerous methodologies for phenotyping root system architecture (RSA), which is the spatial organization of the plant’s root system.Several methods ranging from techniques adapted from medical imaging, such as x-ray tomography (Hargreaves et al., 2008) and combined positron emission tomography-magnetic resonance imaging (Jahnke et al., 2009), to refined versions of classical methods, such as field excavations (Trachsel et al., 2010) and pouch systems (Le Marié et al., 2014), have been used in attempts to understand the phenotypic consequences of genetic and environmental variation on root traits. Each root-phenotyping method has its advantages and disadvantages. Although the medical imaging-based techniques can produce highly detailed representations of roots, they are also very time consuming and require specialized equipment. Excavations, although more easily scaled to higher throughput and not requiring special equipment, are destructive and offer only coarse measurements of RSA. An alternative method for root phenotyping based on an optically clear gel substrate strikes an effective balance between throughput and detail, using a simple digital camera while maintaining precise control over environmental conditions. This platform has been used to quantify and classify distinctive root architectures from 12 rice (Oryza sativa) genotypes (Iyer-Pascuzzi et al., 2010), conduct a quantitative trait locus (QTL) mapping study of rice root traits in three dimensions (Topp et al., 2013), study interspecific and intraspecific rice root interactions (Fang et al., 2013), and quantify contributions of different root types to overall RSA (Clark et al., 2011).Here, we describe the adaptation of this gel imaging platform for use with the large maize root system. We used the platform to quantify the phenotypic diversity of RSA among 25 of the 26 nested association mapping (NAM) founder lines, which encompass a wide spectrum of maize genetic diversity (Yu et al., 2008; McMullen et al., 2009). We found that these lines exhibit diverse RSAs, ranging from small and compact to large and exploratory, suggesting tradeoffs between different types of architectures. In order to identify genetic loci that control maize RSA traits, we characterized a subpopulation that best represented the contrast between the compact and exploratory RSAs. We phenotyped the B73 (compact) × Ki3 (exploratory) recombinant inbred line (RIL) NAM subpopulation for 19 RSA traits at three time points (Topp et al., 2013). These data were used to map 102 QTLs that localized to nine genomic clusters. We found high heritability and large-effect QTLs for most traits, in contrast to maize flowering time QTLs (Buckler et al., 2009). Additionally, several of our QTL clusters overlapped with meta-QTLs for yield traits (Tuberosa et al., 2003; Semagn et al., 2013) as well as novel and previously unreported loci, suggesting that this system can provide a time- and cost-effective means to identify genes controlling root architecture in maize.     

Capturing Arabidopsis Root Architecture Dynamics with root-fit Reveals Diversity in Responses to Salinity

The plant root is the first organ to encounter salinity stress, but the effect of salinity on root system architecture (RSA) remains elusive. Both the reduction in main root (MR) elongation and the redistribution of the root mass between MRs and lateral roots (LRs) are likely to play crucial roles in water extraction efficiency and ion exclusion. To establish which RSA parameters are responsive to salt stress, we performed a detailed time course experiment in which Arabidopsis (Arabidopsis thaliana) seedlings were grown on agar plates under different salt stress conditions. We captured RSA dynamics with quadratic growth functions (root-fit) and summarized the salt-induced differences in RSA dynamics in three growth parameters: MR elongation, average LR elongation, and increase in number of LRs. In the ecotype Columbia-0 accession of Arabidopsis, salt stress affected MR elongation more severely than LR elongation and an increase in LRs, leading to a significantly altered RSA. By quantifying RSA dynamics of 31 different Arabidopsis accessions in control and mild salt stress conditions, different strategies for regulation of MR and LR meristems and root branching were revealed. Different RSA strategies partially correlated with natural variation in abscisic acid sensitivity and different Na⁺/K⁺ ratios in shoots of seedlings grown under mild salt stress. Applying root-fit to describe the dynamics of RSA allowed us to uncover the natural diversity in root morphology and cluster it into four response types that otherwise would have been overlooked.Salt stress is known to affect plant growth and productivity as a result of its osmotic and ionic stress components. Osmotic stress imposed by salinity is thought to act in the early stages of the response, by reducing cell expansion in growing tissues and causing stomatal closure to minimize water loss. The build-up of ions in photosynthetic tissues leads to toxicity in the later stages of salinity stress and can be reduced by limiting sodium transport into the shoot tissue and compartmentalization of sodium ions into the root stele and vacuoles (Munns and Tester, 2008). The effect of salt stress on plant development was studied in terms of ion accumulation, plant survival, and signaling (Munns et al., 2012; Hasegawa, 2013; Pierik and Testerink, 2014). Most studies focus on traits in the aboveground tissues, because minimizing salt accumulation in leaf tissue is crucial for plant survival and its productivity. This approach has led to the discovery of many genes underlying salinity tolerance (Munns and Tester, 2008; Munns et al., 2012; Hasegawa, 2013; Maathuis, 2014). Another way to estimate salinity stress tolerance is by studying the rate of main root (MR) elongation of seedlings transferred to medium supplemented with high salt concentration. This is how Salt Overly Sensitive mutants were identified, being a classical example of genes involved in salt stress signaling and tolerance (Hasegawa, 2013; Maathuis, 2014). The success of this approach is to be explained by the important role that the root plays in salinity tolerance. Roots not only provide anchorage and ensure water and nutrient uptake, but also act as a sensory system, integrating changes in nutrient availability, water content, and salinity to adjust root morphology to exploit available resources to the maximum capacity (Galvan-Ampudia et al., 2013; Gruber et al., 2013). Understanding the significance of environmental modifications of root system architecture (RSA) for plant productivity is one of the major challenges of modern agriculture (de Dorlodot et al., 2007; Den Herder et al., 2010; Pierik and Testerink, 2014).The RSA of dicotyledonous plants consists of an embryonically derived MR and lateral roots (LRs) that originate from xylem pole pericycle cells of the MR, or from LRs in the case of higher-order LRs. Root growth and branching is mainly guided through the antagonistic action of two plant hormones: auxin and cytokinins (Petricka et al., 2012). Under environmental stress conditions, the synthesis of abscisic acid (ABA), ethylene, and brassinosteroids is known to be induced and to modulate the growth of MRs and LRs (Achard et al., 2006; Osmont et al., 2007; Achard and Genschik, 2009; Duan et al., 2013; Geng et al., 2013). In general, lower concentrations of salt were observed to slightly induce MR and LR elongation, whereas higher concentrations resulted in decreased growth of both MRs and LRs (Wang et al., 2009; Zolla et al., 2010). The reduction of growth is a result of the inhibition of cell cycle progression and a reduction in root apical meristem size (West et al., 2004). However, conflicting results were presented for the effect of salinity on lateral root density (LRD; Wang et al., 2009; Zolla et al., 2010; Galvan-Ampudia and Testerink, 2011). Some studies suggest that mild salinity enhances LR initiation or emergence events, thereby affecting patterning, whereas other studies imply that salinity arrests LR development. The origin of those contradictory observations could be attributable to studying LR initiation and density at single time points, rather than observing the dynamics of LR development, because LR formation changes as a function of root growth rate (De Smet et al., 2012). The dynamics of LR growth and development were characterized previously for the MR region formed before the salt stress exposure, identifying the importance of ABA in early growth arrest of postemerged LRs in response to salt stress (Duan et al., 2013). The effect of salt on LR emergence and initiation was found to differ for MR regions formed prior and subsequent to salinity exposure (Duan et al., 2013), consistent with LR patterning being determined at the root tip (Moreno-Risueno et al., 2010). Yet the effect of salt stress on the reprogramming of the entire RSA on a longer timescale remains elusive.Natural variation in Arabidopsis (Arabidopsis thaliana) is a great source for dissecting the genetic components underlying phenotypic diversity (Trontin et al., 2011; Weigel, 2012). Genes underlying phenotypic plasticity of RSA to environmental stimuli were also found to have high allelic variation leading to differences in root development between different Arabidopsis accessions (Rosas et al., 2013). Supposedly, genes responsible for phenotypic plasticity of the root morphology to different environmental conditions are under strong selection for adaptation to local environments. Various populations of Arabidopsis accessions were used to study natural variation in ion accumulation and salinity tolerance (Rus et al., 2006; Jha et al., 2010; Katori et al., 2010; Roy et al., 2013). In addition, a number of studies focusing on the natural variation in RSA have been published, identifying quantitative trait loci and allelic variation for genes involved in RSA development under control conditions (Mouchel et al., 2004; Meijón et al., 2014) and nutrient-deficient conditions (Chevalier et al., 2003; Gujas et al., 2012; Gifford et al., 2013; Kellermeier et al., 2013; Rosas et al., 2013). Exploring natural variation not only expands the knowledge of genes and molecular mechanisms underlying biological processes, but also provides insight on how plants adapt to challenging environmental conditions (Weigel, 2012) and whether the mechanisms are evolutionarily conserved. The early growth arrest of newly emerged LRs upon exposure to salt stress was observed to be conserved among the most commonly used Arabidopsis accessions Columbia-0 (Col-0), Landsberg erecta, and Wassilewskija (Ws; Duan et al., 2013). By studying salt stress responses of the entire RSA and a wider natural variation in root responses to stress, one could identify new morphological traits that are under environmental selection and possibly contribute to stress tolerance.In this work, we not only identify the RSA components that are responsive to salt stress, but we also describe the natural variation in dynamics of salt-induced changes leading to redistribution of root mass and different root morphology. The growth dynamics of MRs and LRs under different salt stress conditions were described by fitting a set of quadratic growth functions (root-fit) to individual RSA components. Studying salt-induced changes in RSA dynamics of 31 Arabidopsis accessions revealed four major strategies conserved among the accessions. Those four strategies were due to differences in salt stress sensitivity of individual RSA components (i.e. growth rates of MRs and LRs, and increases in the number of emerged LRs). This diversity in root morphology responses caused by salt stress was observed to be partially associated with differences in ABA, but not ethylene sensitivity. In addition, we observed that a number of accessions exhibiting a relatively strong inhibition of LR elongation showed a smaller increase in the Na⁺/K⁺ ratio in shoot tissue after exposure to salt stress. Our results imply that different RSA strategies identified in this study reflect diverse adaptations to different soil conditions and thus might contribute to efficient water extraction and ion compartmentalization in their native environments.     

Focus Issue on Roots: Root Nutrient Foraging

During a plant''s lifecycle, the availability of nutrients in the soil is mostly heterogeneous in space and time. Plants are able to adapt to nutrient shortage or localized nutrient availability by altering their root system architecture to efficiently explore soil zones containing the limited nutrient. It has been shown that the deficiency of different nutrients induces root architectural and morphological changes that are, at least to some extent, nutrient specific. Here, we highlight what is known about the importance of individual root system components for nutrient acquisition and how developmental and physiological responses can be coupled to increase nutrient foraging by roots. In addition, we review prominent molecular mechanisms involved in altering the root system in response to local nutrient availability or to the plant''s nutritional status.In natural and agricultural soils, the ability of plants to quickly and efficiently acquire nutrients may determine their competitive success and productivity. Because mineral elements interact differently with themselves and other soil constituents or are carried by water out of the rooted soil volume, their availability to plants may decrease and lead to nutrient deficiency. Under these conditions, plants activate foraging responses that include morphological changes, such as the modulation of root system architecture (RSA) or root hair formation, and physiological changes, such as the release of nutrient-mobilizing root exudates or the expression of nutrient transporters (Gojon et al., 2009; Hinsinger et al., 2009; Gruber et al., 2013). These responses are often spatially coupled to increase the root-soil interaction zone and improve the ability of the plant to intercept immobile nutrients. Noteworthy, although not discussed herein, symbiosis or associative rhizosphere microorganisms can also alter the RSA and enhance the foraging capacity of the plant (Gutjahr and Paszkowski, 2013). Here, we provide an update on the morphological responses induced by plants to forage sparingly available nutrients and some of the underlying molecular mechanisms known to date to be involved in RSA adaptations to nutrient availabilities.     

Focus Issue on Roots: Low Crown Root Number Enhances Nitrogen Acquisition from Low-Nitrogen Soils in Maize

In developing nations, low soil nitrogen (N) availability is a primary limitation to crop production and food security, while in rich nations, intensive N fertilization is a primary economic, energy, and environmental cost to crop production. It has been proposed that genetic variation for root architectural and anatomical traits enhancing the exploitation of deep soil strata could be deployed to develop crops with greater N acquisition. Here, we provide evidence that maize (Zea mays) genotypes with few crown roots (crown root number [CN]) have greater N acquisition from low-N soils. Maize genotypes differed in their CN response to N limitation in greenhouse mesocosms and in the field. Low-CN genotypes had 45% greater rooting depth in low-N soils than high-CN genotypes. Deep injection of ¹⁵N-labeled nitrate showed that low-CN genotypes under low-N conditions acquired more N from deep soil strata than high-CN genotypes, resulting in greater photosynthesis and plant N content. Under low N, low-CN genotypes had greater biomass than high-CN genotypes at flowering (85% in the field study in the United States and 25% in South Africa). In the field in the United States, 1.8× variation in CN was associated with 1.8× variation in yield reduction by N limitation. Our results indicate that CN deserves consideration as a potential trait for genetic improvement of N acquisition from low-N soils.Maize (Zea mays) is one of the world’s most important crops and is a staple food in Latin America and Africa. Maize production requires a large amount of fertilizer, especially nitrogen (N). In the United States, N fertilizers represent the greatest economic and energy costs for maize production (Ribaudo et al., 2011). However, on-farm studies across the northcentral United States revealed that more than half of applied N is not taken up by maize plants and is vulnerable to losses from volatilization, denitrification, and leaching, which pollute air and water resources (Cassman et al., 2002). Conversely, in developing countries, suboptimal N availability is a primary limitation to crop yields and, therefore, food security (Azeez et al., 2006). Increasing yield in these areas is an urgent concern, since chemical fertilizers are not affordable (Worku et al., 2007). Cultivars with greater N acquisition from low-N soils could help alleviate food insecurity in poor nations as well as reduce environmental degradation from excessive fertilizer use in developed countries.The two major soil N forms available to plants are ammonium and nitrate. Nitrate is the main N form in most maize production environments (Miller and Cramer, 2004). Nitrate is highly mobile in soil, and the spatiotemporal availability of soil N is rather complex. In the simplest case, N fertilizers applied to the soil and/or N released from the mineralization of soil organic matter are rapidly converted to nitrate by soil microbes. After irrigation and precipitation events, nitrate moves with water to deeper soil strata. Leaching of nitrate from the root zone has been shown to be a significant cause of low recovery of N fertilizer in commercial agricultural systems (Raun and Johnson, 1999; Cassman et al., 2002). Differences in root depth influence the ability of plants to acquire N. Studies using ¹⁵N-labeled nitrate placed at different soil depths showed that only plants with deep rooting can acquire N sources from deep soil strata, which would otherwise have been lost through leaching (Kristensen and Thorup-Kristensen, 2004a, 2004b). Therefore, selection for root traits enhancing rapid deep soil exploration could be used as a strategy to improve crop N efficiency.The maize root system consists of embryonic and postembryonic components. The embryonic root system consists of two distinct root classes: a primary root and a variable number of seminal roots formed at the scutellar node. The postembryonic root system consists of roots that are formed at consecutive shoot nodes and lateral roots, which are initiated in the pericycle of all root classes. Shoot-borne or nodal roots that are formed belowground are called crown roots, whereas those that are formed aboveground are designated brace roots (Hochholdinger, 2009). While the primary root and seminal roots are essential for the establishment of seedlings after germination, nodal roots and particularly crown roots make up most of the maize root system and are primarily responsible for soil resource acquisition later in development (Hoppe et al., 1986).Lynch (2013) proposed an ideotype for superior N and water acquisition in maize called Steep, Cheap, and Deep (SCD), which integrates root architectural, anatomical, and physiological traits to increase rooting depth and, therefore, the capture of N in leaching environments. One such trait is crown root number (CN). CN is an aggregate trait consisting of the number of belowground nodal whorls and the number of roots per whorl. The crown root system dominates resource acquisition during vegetative growth after the first few weeks and remains important during reproductive development (Hochholdinger et al., 2004). CN in maize ranges from five to 50 under fertile conditions (Trachsel et al., 2011). At the low end of this range, crown roots may be too spatially dispersed to sufficiently explore the soil. There is also a risk of root loss to herbivores and pathogens. If roots are lost in low-N plants, there may be too few crown roots left to support the nutrient, water, and anchorage needs of the plant. At the high end, a large number of crown roots may compete with each other for water and nutrients as well as incur considerable metabolic costs for the plant (Fig. 1). The SCD ideotype proposes that there is an optimal CN for N capture in maize (Lynch, 2013). Under low-N conditions, resources for root growth and maintenance are limiting, and nitrate is a mobile resource that can be captured by a dispersed root system. The optimal CN should tend toward the low end of the phenotypic variation to make resources available for the development of longer, deeper roots rather than more crown roots. According to the SCD ideotype, in low-N soils, maize genotypes with fewer crown roots could explore soils at greater depth, resulting in greater N acquisition, growth, and yield than genotypes with many crown roots.Open in a separate windowFigure 1.Visualization of the maize root system of low- and high-CN genotypes at 40 d after germination. Crown roots are colored in blue, and seminal roots are in red. The CN is eight in the low-CN genotype and 46 in the high-CN genotype. (Image courtesy of Larry M. York.)The objective of this study was to test the hypotheses that (1) low-CN genotypes have greater rooting depth than high-CN genotypes in low-N soils; (2) low-CN genotypes are better at acquiring deep soil N than high-CN genotypes; and (3) low-CN genotypes have greater biomass and yield than high-CN genotypes in low-N conditions.     

Reciprocal Control of Anaplerotic Phosphoenolpyruvate Carboxylase by in Vivo Monoubiquitination and Phosphorylation in Developing Proteoid Roots of Phosphate-Deficient Harsh Hakea

Accumulating evidence indicates important functions for phosphoenolpyruvate (PEP) carboxylase (PEPC) in inorganic phosphate (Pi)-starved plants. This includes controlling the production of organic acid anions (malate, citrate) that are excreted in copious amounts by proteoid roots of nonmycorrhizal species such as harsh hakea (Hakea prostrata). This, in turn, enhances the bioavailability of mineral-bound Pi by solubilizing Al³⁺, Fe³⁺, and Ca²⁺ phosphates in the rhizosphere. Harsh hakea thrives in the nutrient-impoverished, ancient soils of southwestern Australia. Proteoid roots from Pi-starved harsh hakea were analyzed over 20 d of development to correlate changes in malate and citrate exudation with PEPC activity, posttranslational modifications (inhibitory monoubiquitination versus activatory phosphorylation), and kinetic/allosteric properties. Immature proteoid roots contained an equivalent ratio of monoubiquitinated 110-kD and phosphorylated 107-kD PEPC polypeptides (p110 and p107, respectively). PEPC purification, immunoblotting, and mass spectrometry indicated that p110 and p107 are subunits of a 430-kD heterotetramer and that they both originate from the same plant-type PEPC gene. Incubation with a deubiquitinating enzyme converted the p110:p107 PEPC heterotetramer of immature proteoid roots into a p107 homotetramer while significantly increasing the enzyme’s activity under suboptimal but physiologically relevant assay conditions. Proteoid root maturation was paralleled by PEPC activation (e.g. reduced K_m [PEP] coupled with elevated I₅₀ [malate and Asp] values) via in vivo deubiquitination of p110 to p107, and subsequent phosphorylation of the deubiquitinated subunits. This novel mechanism of posttranslational control is hypothesized to contribute to the massive synthesis and excretion of organic acid anions that dominates the carbon metabolism of the mature proteoid roots.Phosphoenolpyruvate (PEP) carboxylase (PEPC; EC 4.1.1.31) is a ubiquitous and tightly regulated cytosolic enzyme of vascular plants that is also widely distributed in green algae and bacteria. PEPC catalyzes the irreversible β-carboxylation of PEP to form oxaloacetate (OAA) and inorganic phosphate (Pi). Vascular plant PEPCs belong to a small multigene family encoding several closely related plant-type PEPCs (PTPCs), along with a distantly related bacterial-type PEPC (BTPC; O’Leary et al., 2011a). PTPC genes encode 105- to 110-kD polypeptides that typically assemble as approximate 400-kD Class-1 PEPC homotetramers. In contrast, BTPC genes encode larger 116- to 118-kD polypeptides owing to a unique intrinsically disordered region that mediates BTPC’s tight interaction with coexpressed PTPC subunits. This association results in the formation of unusual Class-2 PEPC heterooctameric complexes that are largely desensitized to allosteric effectors and that dynamically associate with the surface of mitochondria in vivo (O’Leary et al., 2009, 2011a; Igawa et al., 2010; Park et al., 2012).The critical role of Class-1 PEPC in assimilating atmospheric CO₂ during C₄ and Crassulacean acid metabolism photosynthesis has been studied extensively. Class-1 PEPCs also fulfill a wide range of crucial nonphotosynthetic functions, particularly the anaplerotic replenishment of tricarboxylic acid cycle intermediates consumed during biosynthesis (O’Leary et al., 2011a). Class-1 PEPCs are subject to a complex set of posttranslational controls including allosteric effectors, covalent modification via phosphorylation or monoubiquitination, and protein-protein interactions (Uhrig et al., 2008; O’Leary et al., 2009, 2011a, 2011b). Allosteric activation by Glc-6-P and inhibition by l-malate are routinely observed, whereas phosphorylation and dephosphorylation are catalyzed by a Ca²⁺-independent PEPC protein kinase (PPCK) and a protein phosphatase type-2A (PP2A), respectively (O’Leary et al., 2011a). Phosphorylation at a conserved N-terminal seryl residue activates Class-1 PEPCs by decreasing inhibition by malate while increasing activation by Glc-6-P. By contrast, Class-1 PEPC is subject to inhibitory monoubiquitination during castor oil (Ricinus communis) seed (COS) germination, or following depodding of developing COS (Uhrig et al., 2008; O’Leary et al., 2011b). Immunoblots of germinating COS extracts revealed a 1:1 ratio of immunoreactive 110- and 107-kD PTPC polypeptides (p110 and p107, respectively). PEPC purification and mass spectrometry (MS) demonstrated that (1) p110 and p107 are subunits of a 440-kD Class-1 PEPC heterotetramer, (2) both subunits arise from the same PTPC gene (RcPpc3) that also encodes the phosphorylated 410-kD Class-1 PEPC homotetramer of intact developing COS, and (3) p110 is a monoubiquitinated form of p107 (Uhrig et al., 2008). The monoubiquitination site (Lys-628) of COS p110 is conserved in vascular plant PEPCs and is proximal to a PEP-binding/catalytic domain. Incubation with a deubiquitinating enzyme converted the Class-1 PEPC p110:p107 heterotetramer into a p107 homotetramer while exerting significant effects on the enzyme’s kinetic properties (Uhrig et al., 2008). PTPC monoubiquitination rather than phosphorylation is widespread throughout the astor plant and appears to be the predominant posttranslational modification (PTM) of Class-1 PEPC that occurs in unstressed plants (O’Leary et al., 2011b). The distinctive developmental patterns of Class-1 PEPC phosphoactivation versus monoubiquitination-inhibition indicated that these PTMs might be mutually exclusive in the castor plant (O’Leary et al., 2011a, 2011b).Substantial evidence indicates that PEPC plays a pivotal role in plant acclimation to nutritional Pi deficiency (Duff et al., 1989; Vance et al., 2003; O’Leary et al., 2011a; Plaxton and Tran, 2011; Supplemental Fig. S1), a common abiotic stress that frequently limits plant growth in natural ecosystems. The marked induction of Class-1 PEPCs during Pi stress has been linked to the synthesis and excretion of large amounts of organic acid anions by roots of Pi-starved (–Pi) plants (O’Leary et al., 2011a; Uhde-Stone et al., 2003; Vance et al., 2003; Shane et al., 2004a). The excreted organic acids chelate metal cations such as Al³⁺ and Ca²⁺ that immobilize Pi in the soil, thus increasing soluble Pi concentrations by up to 1,000-fold (Vance et al., 2003). Harsh hakea (Hakea prostrata) is a perennial nonmycotroph that has evolved a host of traits that allow it to thrive in the nutrient-impoverished, ancient soils of western Australia. A crucial adaptation of harsh hakea is its proteoid roots, which excrete copious quantities of citrate and malate to mediate Pi solubilization and acquisition from the soil’s mineral-bound Pi (Supplemental Figs. S1 and S2; Shane et al., 2003, 2004a, 2004b; Shane and Lambers, 2005). Shane and coworkers (2004a) correlated proteoid root development in –Pi harsh hakea with marked increases in respiration, internal carboxylate concentrations, and rates of carboxylate exudation. Immunoblotting indicated that PEPC abundance remained relatively constant during proteoid root development, except in senescing 3-week-old roots, where it showed a marked decline. The PEPC immunoblots also revealed approximately 110- and 100-kD immunoreactive polypeptides that were of equal intensity in young proteoid roots, whereas mature proteoid roots showed a marked reduction in the p110 (Shane et al., 2004a). The possible contribution of PTMs such as phosphorylation to the in vivo activation of proteoid root PEPCs is currently unclear (e.g. see Uhde-Stone et al., 2003). However, this is feasible since the pronounced induction of PPCK genes coupled with the reversible phosphorylation-activation of a Class-1 PEPC isozyme (AtPPC1) has been conclusively demonstrated in –Pi Arabidopsis (Arabidopsis thaliana) suspension cells and seedlings (Gregory et al., 2009).The goal of the current study was to test the hypothesis that PEPC PTMs contribute to the metabolic adaptations of harsh hakea proteoid roots. We report a novel metabolic control paradigm that involves the in vivo deubiquitination and consequent kinetic activation of a phosphorylated form of a C₃ plant Class-1 PEPC.     

Identification of a Hydrolyzable Tannin,Oenothein B,as an Aluminum-Detoxifying Ligand in a Highly Aluminum-Resistant Tree,Eucalyptus camaldulensis

Eucalyptus camaldulensis is a tree species in the Myrtaceae that exhibits extremely high resistance to aluminum (Al). To explore a novel mechanism of Al resistance in plants, we examined the Al-binding ligands in roots and their role in Al resistance of E. camaldulensis. We identified a novel type of Al-binding ligand, oenothein B, which is a dimeric hydrolyzable tannin with many adjacent phenolic hydroxyl groups. Oenothein B was isolated from root extracts of E. camaldulensis by reverse-phase high-performance liquid chromatography and identified by nuclear magnetic resonance and mass spectrometry analyses. Oenothein B formed water-soluble or -insoluble complexes with Al depending on the ratio of oenothein B to Al and could bind at least four Al ions per molecule. In a bioassay using Arabidopsis (Arabidopsis thaliana), Al-induced inhibition of root elongation was completely alleviated by treatment with exogenous oenothein B, which indicated the capability of oenothein B to detoxify Al. In roots of E. camaldulensis, Al exposure enhanced the accumulation of oenothein B, especially in EDTA-extractable forms, which likely formed complexes with Al. Oenothein B was localized mostly in the root symplast, in which a considerable amount of Al accumulated. In contrast, oenothein B was not detected in three Al-sensitive species, comprising the Myrtaceae tree Melaleuca bracteata, Populus nigra, and Arabidopsis. Oenothein B content in roots of five tree species was correlated with their Al resistance. Taken together, these results suggest that internal detoxification of Al by the formation of complexes with oenothein B in roots likely contributes to the high Al resistance of E. camaldulensis.Aluminum (Al) toxicity is a major factor that limits plant growth in acid soils and affects approximately 30% of the total ice-free land area of the world (von Uexküll and Mutert, 1995). Although Al in soils exist in nonphytotoxic silicate or oxide forms at neutral pH, it is solubilized into a phytotoxic form, mainly as Al³⁺, at a pH of less than 5 (Kinraide, 1991; Kochian, 1995). The accumulation of Al in root tips causes rapid inhibition of root elongation, which is a characteristic symptom of Al toxicity in plants (Delhaize and Ryan, 1995; Ma, 2007). In general, plants exhibit an inhibition of root elongation as early as 30 to 120 min after exposure to excessive Al (Barceló and Poschenrieder, 2002). Inhibition of root elongation leads to decreased water and nutrient uptake and, eventually, to restriction of growth of the whole plant.Plants have evolved different levels of Al resistance mediated by two distinct classes of mechanisms (Kochian et al., 2004; Ma, 2007). One strategy is the exclusion of Al from the root tips (exclusion mechanism), and the other is tolerance to Al that enters the root tips (internal tolerance mechanism). The secretion of organic acid anions from roots in response to exposure to Al is the best-documented mechanism for Al exclusion. Organic acid anions (i.e. malate, citrate, and oxalate) can form a complex with Al in the rhizosphere and thereby prevent Al from entering the root tips. The genes encoding transporters for the Al-induced secretion of malate and citrate have been identified and characterized in several plant species (Ryan et al., 2011; Delhaize et al., 2012). Organic acid anions also play a role in the detoxification of Al that enters the roots by means of internal formation of complexes with Al (Ma et al., 1998). However, findings in recent studies increasingly suggest that the Al resistance of some plant species and cultivars cannot be explained solely by these two functions of organic acid anions (Wenzl et al., 2001, 2002; Piñeros et al., 2005; Zheng et al., 2005; Famoso et al., 2010). In addition to organic acid anions, flavonoid-type phenolics (Kidd et al., 2001), phenolic compounds (Ofei-Manu et al., 2001), cyclic hydroxamates (Poschenrieder et al., 2005), and proanthocyanidins (Osawa et al., 2011) in roots or root exudates are proposed as potential organic ligands for Al. The mechanisms by which these additional ligands confer Al resistance remain poorly understood.Eucalyptus camaldulensis is an evergreen tree belonging to the Myrtaceae family and is cultivated in tropical and subtropical regions of the world on account of its superior growth, broad adaptability, and multipurpose wood properties. E. camaldulensis can grow in acid soils and even in acid sulfate soils, where the pH is often lower than 3.5 and the Al concentration in the soil solution often reaches the millimolar level (van Breemen and Pons, 1978). Indeed, seedlings of this species show no inhibition of root elongation and plant growth when exposed to 1 mm Al for 20 d under hydroponic conditions (Tahara et al., 2005). Such Al resistance is considerably higher than that reported for a variety of herbaceous crops and model plants in studies of Al resistance mechanisms; such plants exhibit an inhibition of root elongation at 1 to 50 μm Al (Wenzl et al., 2001). Although our understanding of Al resistance mechanisms in some crops and model plants has improved recently, that for extremely Al-resistant species such as E. camaldulensis is limited.In E. camaldulensis, citrate secretion from roots and its content in the root tips are increased by exposure to Al, suggesting that citrate may contribute to its Al resistance (Tahara et al., 2008a). However, the amounts of organic acid anions, including citrate, secreted from roots and contained within the root tips are lower than those of more sensitive species (Tahara et al., 2008a). Therefore, the high Al resistance of E. camaldulensis cannot be explained only by the presence of organic acid anions. Roots of E. camaldulensis can accumulate large amounts of Al (11 mg g⁻¹ dry weight) with no symptoms of Al toxicity (Tahara et al., 2005), suggesting the existence of additional mechanisms for internal tolerance. In this study, we investigated the presence of novel Al-binding ligands other than organic acid anions in E. camaldulensis roots and identified a hydrolyzable tannin, oenothein B, as a novel type of Al-binding ligand. We also examined the role of the ligand in the internal Al tolerance of E. camaldulensis.     

Ethylene Responses in Rice Roots and Coleoptiles Are Differentially Regulated by a Carotenoid Isomerase-Mediated Abscisic Acid Pathway

Ethylene and abscisic acid (ABA) act synergistically or antagonistically to regulate plant growth and development. ABA is derived from the carotenoid biosynthesis pathway. Here, we analyzed the interplay among ethylene, carotenoid biogenesis, and ABA in rice (Oryza sativa) using the rice ethylene response mutant mhz5, which displays a reduced ethylene response in roots but an enhanced ethylene response in coleoptiles. We found that MHZ5 encodes a carotenoid isomerase and that the mutation in mhz5 blocks carotenoid biosynthesis, reduces ABA accumulation, and promotes ethylene production in etiolated seedlings. ABA can largely rescue the ethylene response of the mhz5 mutant. Ethylene induces MHZ5 expression, the production of neoxanthin, an ABA biosynthesis precursor, and ABA accumulation in roots. MHZ5 overexpression results in enhanced ethylene sensitivity in roots and reduced ethylene sensitivity in coleoptiles. Mutation or overexpression of MHZ5 also alters the expression of ethylene-responsive genes. Genetic studies revealed that the MHZ5-mediated ABA pathway acts downstream of ethylene signaling to inhibit root growth. The MHZ5-mediated ABA pathway likely acts upstream but negatively regulates ethylene signaling to control coleoptile growth. Our study reveals novel interactions among ethylene, carotenogenesis, and ABA and provides insight into improvements in agronomic traits and adaptive growth through the manipulation of these pathways in rice.     

Focus Issue on Roots: Root Cortical Aerenchyma Enhances Nitrogen Acquisition from Low-Nitrogen Soils in Maize

Suboptimal nitrogen (N) availability is a primary constraint for crop production in developing nations, while in rich nations, intensive N fertilization carries substantial environmental and economic costs. Therefore, understanding root phenes that enhance N acquisition is of considerable importance. Structural-functional modeling predicts that root cortical aerenchyma (RCA) could improve N acquisition in maize (Zea mays). We evaluated the utility of RCA for N acquisition by physiological comparison of maize recombinant inbred lines contrasting in RCA grown under suboptimal and adequate N availability in greenhouse mesocosms and in the field in the United States and South Africa. N stress increased RCA formation by 200% in mesocosms and by 90% to 100% in the field. RCA formation substantially reduced root respiration and root N content. Under low-N conditions, RCA formation increased rooting depth by 15% to 31%, increased leaf N content by 28% to 81%, increased leaf chlorophyll content by 22%, increased leaf CO₂ assimilation by 22%, increased vegetative biomass by 31% to 66%, and increased grain yield by 58%. Our results are consistent with the hypothesis that RCA improves plant growth under N-limiting conditions by decreasing root metabolic costs, thereby enhancing soil exploration and N acquisition in deep soil strata. Although potential fitness tradeoffs of RCA formation are poorly understood, increased RCA formation appears be a promising breeding target for enhancing crop N acquisition.Nitrogen (N) deficiency is one of the most limiting factors in maize (Zea mays) production worldwide (Ladha et al., 2005). In developing countries such as those in sub-Saharan Africa, less than 20 kg N ha⁻¹ is applied to fields of smallholder farmers due to high fertilizer cost (Azeez et al., 2006; Worku et al., 2007). In developed countries, intensive N fertilization is used to maintain satisfactory yield (Tilman et al., 2002). In the United States, N fertilizers are the greatest economic and energy cost for maize production (Ribaudo et al., 2011). However, less than half of the N applied to crops is actually acquired, and most of the remaining N becomes a source of environmental pollution (Raun and Johnson, 1999; Smil, 1999; Tilman et al., 2002). For example, N and phosphorus (P) effluents into marine systems from agriculture cause eutrophication and hypoxic zones (Diaz and Rosenberg, 2008; Robertson and Vitousek, 2009). Nitrate contamination in surface water and groundwater systems poses serious health risks, such as methemoglobinemia and N-nitroso-induced cancers (UNEP and WHRC, 2007). Emission of nitrous oxides from agricultural activities contributes to ozone damage and global warming (Kulkarni et al., 2008; Sutton et al., 2011). Furthermore, the production of N fertilizers requires considerable energy from fossil fuels, and since energy costs have risen in recent years, farmers face economic pressure from increasing N fertilizer costs, which are linked to higher food prices. It is estimated that a 1% increase in crop N efficiency could save more than $1 billion (U.S.) annually worldwide (Kant et al., 2011). Therefore, even a small improvement in N efficiency would have significant positive impacts on the environment and the economy.Soil N is heterogenous and dynamic. The bioavailability of soil N depends on the balance between the rates of mineralization, nitrification, and denitrification. These processes are determined by several factors, including soil composition, microbial activity, soil temperature, and soil water status (Miller and Cramer, 2004). The predominant form of soil N available to plants in most agricultural systems is nitrate, which is highly soluble in water and thus mobile in the soil (Barber, 1995; Marschner, 1995). Mineralization of organic matter and/or the application of N fertilizer at the beginning of the growing season followed by precipitation and irrigation create a pulse of nitrate that may exceed the N acquisition capacity of seedlings and leach below the root zone. Therefore, it has been proposed that increasing the speed of root exploration of deep soil strata could benefit N acquisition (Lynch, 2013). However, the structural investments and metabolic expenditures of root systems are substantial and can exceed half of daily photosynthesis (Lambers et al., 2002). Therefore, full consideration of the costs and benefits of root systems is crucial for identifying root traits to improve crop production, especially in water- and nutrient-deficient environments (Lynch, 2007). Taking rhizoeconomics and the spatiotemporal availability of soil N into account, Lynch (2013) proposed a root ideotype for enhanced N acquisition in maize called Steep, Cheap, and Deep, in which Steep refers to architectural phenes and Cheap refers to phenes that reduce the metabolic cost of soil exploration. One element of this ideotype is abundant root cortical aerenchyma (RCA).RCA consists of enlarged air spaces in the root cortex (Esau, 1977). RCA is known to form in response to hypoxia, and the role of RCA in improving oxygen transport to roots of many plant species under hypoxic conditions has been well researched (Vartapetian and Jackson, 1997; Jackson and Armstrong, 1999; Mano and Omori, 2007, 2013). Interestingly, RCA can also form in response to drought and edaphic stresses such as N, P, and sulfur deficiencies (Drew et al., 1989; Bouranis et al., 2003; Fan et al., 2003; Zhu et al., 2010a), which suggests that the benefit of RCA extends beyond facilitating oxygen transport. Several lines of evidence suggest that RCA enhances root metabolic efficiency under stress. Fan et al. (2003) found that RCA formation significantly reduced root segment respiration and P content of root tissue, which allowed greater shoot growth in soils with low P availability. Under drought, maize genotypes with high RCA formation had greater root length, deeper rooting, better leaf water status, and 8 times greater yield than closely related genotypes with low RCA (Zhu et al., 2010a). Effects of RCA on root respiration were more pronounced for large-diameter roots compared with small-diameter roots (Jaramillo et al., 2013). Results from the functional-structural plant model SimRoot showed that RCA formation could be an adaptive response to deficiency of N, P, and potassium by decreasing the metabolic cost of soil exploration. By reducing root respiration, RCA decreases the carbon cost of soil exploration, and by decreasing the N and P content of root tissue, RCA permits internal reallocation of nutrients to growing root tissue, which is particularly beneficial under conditions of low N and P availability (Postma and Lynch, 2011a). Under suboptimal P availability, RCA increased the growth of a simulated 40-d-old maize plant by 70% (Postma and Lynch, 2011b). In the case of N, RCA increased the growth of simulated maize plants up to 55% in low-N conditions, and plants benefit from RCA more in high-N-leaching environments than in low-N-leaching environments (Postma and Lynch, 2011a). In addition, the formation of RCA decreases critical soil nutrient levels, defined as the soil fertility below which growth is reduced, suggesting that cultivars with high RCA may require less fertilizer under nonstressed conditions. These in silico results suggest that RCA has potential utility for improving crop nutrient acquisition in both high- and low-input agroecosystems.The overall objective of this research was to assess the utility of RCA for N acquisition in maize under N-limiting conditions. Maize near-isophenic recombinant inbred lines (RILs) sharing a common genetic background (i.e. descending from the same parents) with common root phenotypes but contrasting in RCA formation were grown under N stress to test the hypothesis that RCA formation is associated with reduced root respiration, reduced tissue nutrient content, greater rooting depth, enhanced N acquisition, and therefore greater plant growth and yield under N limitation.     

In Situ Speciation and Distribution of Toxic Selenium in Hydrated Roots of Cowpea

The speciation and spatial distribution of selenium (Se) in hydrated plant tissues is not well understood. Using synchrotron-based x-ray absorption spectroscopy and x-ray fluorescence microscopy (two-dimensional scanning [and associated mathematical model] and computed tomography), the speciation and distribution of toxic Se were examined within hydrated roots of cowpea (Vigna unguiculata) exposed to either 20 µm selenite or selenate. Based upon bulk solution concentrations, selenate was 9-fold more toxic to the roots than selenite, most likely due to increased accumulation of organoselenium (e.g. selenomethionine) in selenate-treated roots. Specifically, uptake of selenate (probably by sulfate transporters) occurred at a much higher rate than for selenite (apparently by both passive diffusion and phosphate transporters), with bulk root tissue Se concentrations approximately 18-fold higher in the selenate treatment. Although the proportion of Se converted to organic forms was higher for selenite (100%) than for selenate (26%), the absolute concentration of organoselenium was actually approximately 5-fold higher for selenate-treated roots. In addition, the longitudinal and radial distribution of Se in roots differed markedly: the highest tissue concentrations were in the endodermis and cortex approximately 4 mm or more behind the apex when exposed to selenate but in the meristem (approximately 1 mm from the apex) when exposed to selenite. The examination of the distribution and speciation of Se in hydrated roots provides valuable data in understanding Se uptake, transport, and toxicity.Selenium (Se) is an essential micronutrient for humans and other animals (Rayman, 2008). At elevated concentrations, however, it is toxic, and the concentration range from deficiency to lethality is unusually narrow (Terry et al., 2000). Plants represent a direct entrance to the wider food chain as the main sources of dietary Se (Rayman, 2008). The uptake and accumulation of Se by plants is an important process in controlling the health risks resulting from Se deficiency or toxicity. Se toxicity to plants has been observed in arid and semiarid soils derived from seleniferous rocks and shales, although anthropogenic contamination is also of concern (Terry et al., 2000). Therefore, it is important that the mechanisms of Se uptake, transformation, and toxicity in plants are understood in order to reduce health risks.Selenite (Se[IV]) and selenate (Se[VI]) are the two dominant inorganic species in soils depending upon the redox potential and pH (Elrashidi et al., 1987). The mechanism of Se[VI] uptake is well known: it is taken up by plant roots via the high-affinity sulfate transporters (Terry et al., 2000) due to the similarity between Se[VI] and sulfate. By contrast, little is known about the uptake mechanism involved in Se[IV] in plant roots. Some studies suggested that Se[IV] is taken up via passive diffusion (Shrift and Ulrich, 1969; Arvy, 1993). Recently, Zhao et al. (2010) reported that the uptake of Se[IV] is mediated by the silicon (Si) influx transporter Lsi1 (OsNIP2;1) in rice (Oryza sativa). Furthermore, Se[IV] uptake was found to occur via both passive diffusion and phosphate transporters in the marine coccolithophore Emiliania huxleyi (Araie et al., 2011). Apart from the difference in their mechanisms of uptake, they also differ in their mobility within plants (Li et al., 2008). Se[VI] is relatively easily translocated from roots to shoots, whereas Se[IV] tends to accumulate within the roots (Arvy, 1993). Despite this important progress, much less is known about the sites of uptake of Se[IV]/Se[VI] and their possible chemical transformations in hydrated plant roots. This information regarding the in situ distribution and chemical forms of Se would be helpful in elucidating the mechanism(s) responsible for Se uptake, transformation, and toxicity in plants.Recent advances in synchrotron-based techniques allow in situ measurement of the distribution of metal(loid)s in hydrated fresh plant tissues (Kopittke et al., 2011, 2012; Lombi et al., 2011a). In particular, the prototype Maia detector system, jointly developed by the Australian Synchrotron, the Commonwealth Scientific and Industrial Research Organization, and the Brookhaven National Laboratory, represents a new-generation x-ray fluorescence detector and real-time processing approach that provides unprecedented capabilities in in situ element imaging and measurement (Lombi et al., 2011b). The Maia uses an annular array of 384 silicon-diode detectors positioned in a backscatter geometry to subtend a large solid angle (approximately 1.3 steradian) and to achieve high count-rate capacity (Kirkham et al., 2010). Data acquisition times are approximately 10 to 100 times faster in the Maia than for other detectors, thereby allowing analysis of highly hydrated biological specimens (e.g. roots) without observable damage (Lombi et al., 2011a). This has allowed us to overcome the analytical challenges of examining the two-dimensional and virtual three-dimensional distribution of low-concentration metal(loid)s in hydrated and fresh plant tissues (Kopittke et al., 2011, 2012; Lombi et al., 2011a, 2011c).In this study, we investigated the speciation and quantified the longitudinal and radial distribution of Se in hydrated roots of cowpea (Vigna unguiculata) exposed to either Se[IV] or Se[VI]. Cowpea is a model species of rhizotoxicity and is also one of the most important food legume crops in the semiarid tropics, where Se toxicity is often a concern. The chemical forms of Se in cowpea roots were first examined using x-ray absorption spectroscopy (XAS). Second, with x-ray fluorescence microscopy (µ-XRF), we used two-dimensional imaging (coupled with an associated mathematical model to calculate concentrations of Se within various tissues of the root cylinder) to determine the spatially resolved distribution of Se within root tissues. Additionally, sequential tomography was used to provide virtual three-dimensional reconstructions of Se distribution in roots, enabling comparison of computed tomography with the mathematical model.     

Salicylic Acid Regulates Arabidopsis Microbial Pattern Receptor Kinase Levels and Signaling

In Arabidopsis thaliana, responses to pathogen-associated molecular patterns (PAMPs) are mediated by cell surface pattern recognition receptors (PRRs) and include the accumulation of reactive oxygen species, callose deposition in the cell wall, and the generation of the signal molecule salicylic acid (SA). SA acts in a positive feedback loop with ACCELERATED CELL DEATH6 (ACD6), a membrane protein that contributes to immunity. This work shows that PRRs associate with and are part of the ACD6/SA feedback loop. ACD6 positively regulates the abundance of several PRRs and affects the responsiveness of plants to two PAMPs. SA accumulation also causes increased levels of PRRs and potentiates the responsiveness of plants to PAMPs. Finally, SA induces PRR- and ACD6-dependent signaling to induce callose deposition independent of the presence of PAMPs. This PAMP-independent effect of SA causes a transient reduction of PRRs and ACD6-dependent reduced responsiveness to PAMPs. Thus, SA has a dynamic effect on the regulation and function of PRRs. Within a few hours, SA signaling promotes defenses and downregulates PRRs, whereas later (within 24 to 48 h) SA signaling upregulates PRRs, and plants are rendered more responsive to PAMPs. These results implicate multiple modes of signaling for PRRs in response to PAMPs and SA.