Auxin Resistant1 and PIN-FORMED2 Protect Lateral Root Formation in Arabidopsis under Iron Stress

A stunted root system is a significant symptom of iron (Fe) toxicity, yet little is known about the effects of excess Fe on lateral root (LR) development. In this work, we show that excess Fe has different effects on LR development in different portions of the Arabidopsis (Arabidopsis thaliana) root system and that inhibitory effects on the LR initiation are only seen in roots newly formed during excess Fe exposure. We show that root tip contact with Fe is both necessary and sufficient for LR inhibition and that the auxin, but not abscisic acid, pathway is engaged centrally in the initial stages of excess Fe exposure. Furthermore, Fe stress significantly reduced PIN-FORMED2 (PIN2)-green fluorescent protein (GFP) expression in root tips, and pin2-1 mutants exhibited significantly fewer LR initiation events under excess Fe than the wild type. Exogenous application of both Fe and glutathione together increased PIN2-GFP expression and the number of LR initiation events compared with Fe treatment alone. The ethylene inhibitor aminoethoxyvinyl-glycine intensified Fe-dependent inhibition of LR formation in the wild type, and this inhibition was significantly reduced in the ethylene overproduction mutant ethylene overproducer1-1. We show that Auxin Resistant1 (AUX1) is a critical component in the mediation of endogenous ethylene effects on LR formation under excess Fe stress. Our findings demonstrate the relationship between excess Fe-dependent PIN2 expression and LR formation and the potential role of AUX1 in ethylene-mediated LR tolerance and suggest that AUX1 and PIN2 protect LR formation in Arabidopsis during the early stages of Fe stress.Iron (Fe) is an essential trace element for plants (Pilon et al., 2009), and species differ greatly in how much Fe they require for optimal growth (Wheeler and Power, 1995; Batty and Younger, 2003). As Fe is frequently limiting, Fe deficiency is more commonly studied than toxicity arising from excess Fe exposure (Lei et al., 2014; Bashir et al., 2015; Briat et al., 2015). Fe is also a major focus for efforts in biofortification by targeting Fe transporters (Zhai et al., 2014; Pinto and Ferreira, 2015). However, the excessive presence of Fe in soils is equally common, in particular in soils characterized by low pH and hypoxic or anoxic conditions (Connolly and Guerinot, 2002). Toxicity arising from excess Fe exposure is recognized as one of the major plant diseases attributable to abiotic factors that impact the development and yield potential in the world’s leading cereal crops, rice (Oryza sativa) and wheat (Triticum aestivum; Becker and Asch, 2005; Khabaz-Saberi et al., 2012). Understanding the mechanisms underlying excess Fe toxicity is therefore essential.Plastic responses in the plant’s root system architecture are known to constitute a major mechanism by which plants cope with fluctuating environments. Lateral roots (LRs), which typically comprise the majority of the root system, contribute pivotally to nutrient acquisition from soil, and modulating LR development is a very important avoidance strategy for plants when confronted with unfavorable edaphic conditions, such as high salinity or heavy metals (Ivanov et al., 2003). In the case of excess exposure to Fe, stunting of the root system is among the chief symptoms of toxicity (Becker and Asch, 2005). However, while some information has been emerging on the primary root axis (Li et al., 2015), the specific role of the plant’s LR apparatus remains poorly studied. Yamauchi and Peng (1995) reported retardation of root growth and a reduction in LR length and number under excess Fe conditions. Recently, Reyt et al. (2015) showed that excess Fe had no significant effect on LR initiation in the LR branching zone and that ferritins play an important role in LR emergence under excess Fe in this portion of the root, although the authors had not investigated LR development in the root portions near the growing tip of the primary root. Because LR initiation is restricted to specific pericycle cell files adjacent to a xylem pole in the basal region of the meristem (De Smet et al., 2007; Fukaki and Tasaka, 2009), and LR formation in this new growing root portion may be more susceptible to stress stimuli, such as observed with exposure to high NH₄⁺ and salt (Duan et al., 2013; Li et al., 2013), it is reasonable to suggest that modulation of LR formation near the growing tip of the primary root is critical to the response to excess Fe stress.In Arabidopsis (Arabidopsis thaliana), the development of LRs proceeds through the following stages: lateral root primordia (LRP) initiation, establishment, emergence, activation into mature LRs, and final maintenance of LR elongation (Fukaki and Tasaka, 2009; Péret et al., 2009). The hormones abscisic acid (ABA) and auxin are important internal negative and positive regulators during LR development, respectively (Fukaki and Tasaka, 2009). ABA has been implicated in LRP emergence and meristem activation independent of auxin (De Smet et al., 2003). Auxin is an important internal positive regulator during LR development (Fukaki and Tasaka, 2009), and auxin transport is critical (Blilou et al., 2005). Mutants in auxin efflux carriers such as PIN-FORMED (PIN) and P-Glycoprotein show significant defects in LR formation (Fukaki and Tasaka, 2009; Péret et al., 2009). For example, LR initiation frequency was significantly reduced in pin2 and pin3 mutants (Dubrovsky et al., 2009), and PIN2 was also shown to be involved in exogenous and endogenous signal-mediated LR development (by brassinosteroid, jasmonate, and fungal challenge; Li et al., 2005; Felten et al., 2009; Sun et al., 2009). Similarly, Auxin Resistant1 (AUX1), an auxin influx carrier, also regulates LRP positioning and initiation (De Smet et al., 2007). While both AUX1 and PIN2 are required specifically for the basipetal transport of auxin through the outer root cell layers (Fukaki and Tasaka, 2009), PIN1 localized at the basal end of vascular cells is responsible for direct acropetal auxin flow in the root stele (Blilou et al., 2005). Recently, the roles of ethylene on LR development have also been highlighted, and the ethylene-mediated LR formation is dependent on the auxin pathway (Ivanchenko et al., 2008; Lewis et al., 2011). Ethylene treatment could mediate fluorescence of AUX1 and PIN2 fluorescent protein fusions at the root tip (Růzicka et al., 2007; Lewis et al., 2011). Although ABA, auxin, and ethylene signals have been implicated as important for LR development, it is not known whether and how the three hormones are involved in the response of LR formation to Fe stress.The previously described phenotypes and physiological processes related to Fe toxicity do not clarify the effect of excess Fe on LR formation. In this study, we employed the Arabidopsis wild type and ABA-, auxin-, and ethylene-related mutants to explore the LR formation response to Fe toxicity and to elucidate the roles of ABA, auxin, and ethylene. Potential mechanisms involved in the early stress response to Fe stress are discussed.     

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.     

Live Imaging of Inorganic Phosphate in Plants with Cellular and Subcellular Resolution

Despite variable and often scarce supplies of inorganic phosphate (Pi) from soils, plants must distribute appropriate amounts of Pi to each cell and subcellular compartment to sustain essential metabolic activities. The ability to monitor Pi dynamics with subcellular resolution in live plants is, therefore, critical for understanding how this essential nutrient is acquired, mobilized, recycled, and stored. Fluorescence indicator protein for inorganic phosphate (FLIPPi) sensors are genetically encoded fluorescence resonance energy transfer-based sensors that have been used to monitor Pi dynamics in cultured animal cells. Here, we present a series of Pi sensors optimized for use in plants. Substitution of the enhanced yellow fluorescent protein component of a FLIPPi sensor with a circularly permuted version of Venus enhanced sensor dynamic range nearly 2.5-fold. The resulting circularly permuted FLIPPi sensor was subjected to a high-efficiency mutagenesis strategy that relied on statistical coupling analysis to identify regions of the protein likely to influence Pi affinity. A series of affinity mutants was selected with dissociation constant values of 0.08 to 11 mm, which span the range for most plant cell compartments. The sensors were expressed in Arabidopsis (Arabidopsis thaliana), and ratiometric imaging was used to monitor cytosolic Pi dynamics in root cells in response to Pi deprivation and resupply. Moreover, plastid-targeted versions of the sensors expressed in the wild type and a mutant lacking the PHOSPHATE TRANSPORT4;2 plastidic Pi transporter confirmed a physiological role for this transporter in Pi export from root plastids. These circularly permuted FLIPPi sensors, therefore, enable detailed analysis of Pi dynamics with subcellular resolution in live plants.Phosphorus is an essential element that plants acquire and assimilate in the form of inorganic phosphate (Pi). This macronutrient is a component of numerous metabolites and macromolecules, including ATP, nucleic acids, and phospholipids, and serves key roles in energy transfer reactions, signal transduction processes, and regulation of enzyme activities. Of fundamental importance to plants, Pi also serves critical roles in photosynthesis as both a substrate for ATP synthesis through photophosphorylation and a regulator in the partitioning of fixed carbon between the starch and Suc biosynthetic pathways.In many soils, particularly those used for low-input agriculture, the amounts of Pi available to plants are limiting for growth and productivity (Vance et al., 2003). Most of the Pi in soils is unavailable, because it is immobilized through formation of insoluble complexes or exists in organic forms, such as phytate, that plants cannot use directly (Schachtman et al., 1998). As a result, concentrations of free Pi in soil solution range from 1 to 10 μm, whereas cells require Pi in the millimolar range (Bieleski, 1973).To acclimate to Pi limitation, plants have evolved mechanisms to enhance Pi acquisition and also, mobilize, recycle, and conserve internal stores. These mechanisms include secretion of organic acids and phosphatases (Vance et al., 2003), increased growth of lateral roots and root hairs (Bates and Lynch, 2000; Péret et al., 2011), production of high-affinity Pi transporters at the root-soil interface (Misson et al., 2004; Shin et al., 2004), formation of symbiotic association with mycorrhizal fungi, which enhances Pi scavenging capabilities (Javot et al., 2007), modification of metabolic pathways (Plaxton and Tran, 2011), and altered patterns of Pi translocation between organs and transport between subcellular compartments (Walker and Sivak, 1986; Mimura, 1999; Raghothama, 1999). Substantial insights have been gained into the underlying biochemical identities and regulatory strategies for such adaptive responses, including those related to sensing and signaling of Pi status (Rouached et al., 2010; Chiou and Lin, 2011; Plaxton and Tran, 2011; Jain et al., 2012; Liu et al., 2014; Zhang et al., 2014). However, a thorough understanding of their respective mechanisms and how these are integrated is limited by the inability to assess intracellular Pi concentrations with high spatial and temporal resolution.Genetically encoded fluorescent sensors or biosensors have proven to be powerful tools for monitoring metabolites and ions in vivo, because their expression and subcellular targeting can be manipulated and fluorescence imaging is nondestructive (Lalonde et al., 2005; Okumoto et al., 2012). Sensor proteins are fusions of a ligand binding domain or protein with one or two fluorescent proteins (e.g. GFP and related variants). Sensors with a single fluorescent protein report ligand-dependent changes in conformation as changes in fluorescence intensity, whereas sensors with two fluorescent proteins can yield changes in fluorescence resonance energy transfer (FRET), which can be quantified through ratiometric imaging. FRET-based sensors have been used in live plants to assess a variety of analytes, including Glc, maltose, Suc, Gln, calcium, zinc, and pH (Deuschle et al., 2006; Chaudhuri et al., 2008, 2011; Kaper et al., 2008; Rincón-Zachary et al., 2010; Adams et al., 2012; Gjetting et al., 2012, 2013; Krebs et al., 2012).Gu et al. (2006) engineered a FRET-based Pi sensor named fluorescence indicator protein for inorganic phosphate (FLIPPi) that consists of a cyanobacterial inorganic phosphate binding protein (PiBP) fused to enhanced cyan fluorescent protein (eCFP) and enhanced yellow fluorescent protein (eYFP) and showed the use of one of these sensors for monitoring cytosolic Pi in cultured animal cells. In this study, we generated a series of second generation FLIPPi sensors that were modified and optimized for use in live plants. Substitution of eYFP with a circularly permuted (cp) form of the fluorescent protein Venus (cpVenus; Nagai et al., 2002, 2004) greatly increased the magnitude of Pi-dependent FRET responses. In keeping with the initial nomenclature, Pi sensors constructed with cpVenus were designated cpFLIPPi. We also used a targeted mutagenesis approach to obtain cpFLIPPi sensors with Pi binding affinities that spanned the physiological range of most cell compartments and expressed these in Arabidopsis (Arabidopsis thaliana). Confocal microscopy coupled with ratiometric analysis or acceptor photobleaching detected changes in cytosolic Pi levels in root epidermal cells in response to Pi starvation, and these changes were fully reversed by Pi replenishment. Plastid-localized versions of the same sensors expressed in wild-type plants and mutants lacking the PHOSPHATE TRANSPORT4;2 (PHT4;2) plastidic Pi transporter (Irigoyen et al., 2011) were used to confirm a role for this transporter in the export of Pi from root plastids. These results show the use of cpFLIPPi sensors for monitoring Pi distributions with both cellular and subcellular resolutions in live plants.