Jasmonic Acid-Ethylene Crosstalk via ETHYLENE INSENSITIVE 2 Reprograms Arabidopsis Root System Architecture Through Nitric Oxide Accumulation

Journal of Plant Growth Regulation - Plant growth and development are tightly regulated by phytohormones, including jasmonic acid (JA) and ethylene (ET), two canonical players in plant defense and...     

植物根构型的定量分析

植物根系具有锚定植株、吸收和运输土壤中的水分及养分、合成和贮藏营养物质等重要功能。根构型是根系在土壤中的空间造型和分布。对植物根构型进行定量分析,有助于人们了解根系结构和根系功能在生态系统中的重要作用。本文对植物根构型的概念及其定量分析研究进展进行了概述,并介绍了植物根构型的主要研究方法和定量分析技术。     

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.     

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.     

Crosstalk between the Circadian Clock and Innate Immunity in Arabidopsis

The circadian clock integrates temporal information with environmental cues in regulating plant development and physiology. Recently, the circadian clock has been shown to affect plant responses to biotic cues. To further examine this role of the circadian clock, we tested disease resistance in mutants disrupted in CCA1 and LHY, which act synergistically to regulate clock activity. We found that cca1 and lhy mutants also synergistically affect basal and resistance gene-mediated defense against Pseudomonas syringae and Hyaloperonospora arabidopsidis. Disrupting the circadian clock caused by overexpression of CCA1 or LHY also resulted in severe susceptibility to P. syringae. We identified a downstream target of CCA1 and LHY, GRP7, a key constituent of a slave oscillator regulated by the circadian clock and previously shown to influence plant defense and stomatal activity. We show that the defense role of CCA1 and LHY against P. syringae is at least partially through circadian control of stomatal aperture but is independent of defense mediated by salicylic acid. Furthermore, we found defense activation by P. syringae infection and treatment with the elicitor flg22 can feedback-regulate clock activity. Together this data strongly supports a direct role of the circadian clock in defense control and reveal for the first time crosstalk between the circadian clock and plant innate immunity.     

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.     

Transcriptional Changes of the Root-Knot Nematode Meloidogyne incognita in Response to Arabidopsis thaliana Root Signals

Root-knot nematodes are obligate parasites that invade roots and induce the formation of specialized feeding structures. Although physiological and molecular changes inside the root leading to feeding site formation have been studied, very little is known about the molecular events preceding root penetration by nematodes. In order to investigate the influence of root exudates on nematode gene expression before plant invasion and to identify new genes potentially involved in parasitism, sterile root exudates from the model plant Arabidopsis thaliana were produced and used to treat Meloidogyne incognita pre-parasitic second-stage juveniles. After confirming the activity of A. thaliana root exudates (ARE) on M. incognita stylet thrusting, six new candidate genes identified by cDNA-AFLP were confirmed by qRT-PCR as being differentially expressed after incubation for one hour with ARE. Using an in vitro inoculation method that focuses on the events preceding the root penetration, we show that five of these genes are differentially expressed within hours of nematode exposure to A. thaliana roots. We also show that these genes are up-regulated post nematode penetration during migration and feeding site initiation. This study demonstrates that preceding root invasion plant-parasitic nematodes are able to perceive root signals and to respond by changing their behaviour and gene expression.     

Natural Variation of Arabidopsis Root Architecture Reveals Complementing Adaptive Strategies to Potassium Starvation

Root architecture is a highly plastic and environmentally responsive trait that enables plants to counteract nutrient scarcities with different foraging strategies. In potassium (K) deficiency (low K), seedlings of the Arabidopsis (Arabidopsis thaliana) reference accession Columbia (Col-0) show a strong reduction of lateral root elongation. To date, it is not clear whether this is a direct consequence of the lack of K as an osmoticum or a triggered response to maintain the growth of other organs under limiting conditions. In this study, we made use of natural variation within Arabidopsis to look for novel root architectural responses to low K. A comprehensive set of 14 differentially responding root parameters were quantified in K-starved and K-replete plants. We identified a phenotypic gradient that links two extreme strategies of morphological adaptation to low K arising from a major tradeoff between main root (MR) and lateral root elongation. Accessions adopting strategy I (e.g. Col-0) maintained MR growth but compromised lateral root elongation, whereas strategy II genotypes (e.g. Catania-1) arrested MR elongation in favor of lateral branching. K resupply and histochemical staining resolved the temporal and spatial patterns of these responses. Quantitative trait locus analysis of K-dependent root architectures within a Col-0 × Catania-1 recombinant inbred line population identified several loci each of which determined a particular subset of root architectural parameters. Our results indicate the existence of genomic hubs in the coordinated control of root growth in stress conditions and provide resources to facilitate the identification of the underlying genes.The ability of plants to actively respond to nutrient scarcity with changes in root architecture is a fascinating phenomenon. Advances in root research and breeding efforts that focus on the enhancement of root traits have been recognized as principal goals to ensure those high yields necessary to feed an ever-growing human population (Hammer et al., 2009; Den Herder et al., 2010). Indeed, understanding the adaptations of root systems to environmental factors has been pointed out as a key issue in modern agriculture (Den Herder et al., 2010).Potassium (K) is the quantitatively most important cation for plant growth, as it serves as the major osmoticum for cell expansion (Leigh and Wyn Jones, 1984; Amtmann et al., 2006). Moreover, K is essential for many cellular and tissue processes, such as enzymatic activity, transport of minerals and metabolites, and regulation of stomatal aperture (Amtmann et al., 2006). Even in fertilized fields, rapid K uptake by plants can lead to K shortage in the root environment, especially early in the growth season. Root adaptations to K deficiency (low K) take place at the physiological (Armengaud et al., 2004; Shin and Schachtman, 2004; Alemán et al., 2011), metabolic (Armengaud et al., 2009a), and morphological levels. In a classic study, Drew (1975) showed an increase in overall lateral root (LR) growth of barley seedlings, even when K was supplied only to parts of the root system. Conversely, a typical response of Arabidopsis (Arabidopsis thaliana) Columbia (Col-0) seedlings to low K is the drastic reduction of LR elongation (Armengaud et al., 2004; Shin and Schachtman, 2004). Conflicting data have been published on the effect of low K on main root (MR) growth in the same species, ranging from no effect (Shin and Schachtman, 2004) to impaired MR elongation (Jung et al., 2009; Kim et al., 2010). Some components involved in K starvation responses have been identified, such as jasmonates (Armengaud et al., 2004, 2010), reactive oxygen species (Shin and Schachtman, 2004), and ethylene (Jung et al., 2009). However, the molecular identity of a root K sensor acting at the base of the signaling cascade is so far unknown.Genetic variation within species is a useful resource to dissect the genetic components determining phenotypes (Koornneef et al., 2004; Trontin et al., 2011; Weigel, 2012). Natural variation within Arabidopsis has been the basis for many studies on plant morphology, physiology, and development as well as stress response (Alonso-Blanco et al., 2009; Weigel, 2012). Natural variation of root traits such as primary root length (Mouchel et al., 2004; Loudet et al., 2005; Sergeeva et al., 2006), LR length (Loudet et al., 2005), and total root size (Fitz Gerald et al., 2006) have pinpointed genomic regions underlying the phenotypic variation via mapping of quantitative trait loci (QTLs) as a first step toward the identification of novel regulatory genes (Mouchel et al., 2004). This strategy has also been applied to environmental responses, such as growth responses to phosphate starvation (Reymond et al., 2006; Svistoonoff et al., 2007). However, despite their importance for plant growth and their strong effect on overall root architecture, root responses to K deficiency have not been genetically dissected.Here, we show that Arabidopsis accessions follow different strategies to adapt to K starvation. We present the quantification of a comprehensive set of root architectural parameters of Arabidopsis grown in K-sufficient and K-deficient media and the identification of genetic loci, each of which determines the response of a distinct subset of root architectural parameters to K starvation.     

Pseudomonas putida and Pseudomonas fluorescens Influence Arabidopsis Root System Architecture Through an Auxin Response Mediated by Bioactive Cyclodipeptides

Journal of Plant Growth Regulation - Plant growth-promoting rhizobacteria modulate root development through different mechanisms. This work was conducted to evaluate the effects of root...     

Kernel Architecture of the Genetic Circuitry of the Arabidopsis Circadian System

A wide range of organisms features molecular machines, circadian clocks, which generate endogenous oscillations with ~24 h periodicity and thereby synchronize biological processes to diurnal environmental fluctuations. Recently, it has become clear that plants harbor more complex gene regulatory circuits within the core circadian clocks than other organisms, inspiring a fundamental question: are all these regulatory interactions between clock genes equally crucial for the establishment and maintenance of circadian rhythms? Our mechanistic simulation for Arabidopsis thaliana demonstrates that at least half of the total regulatory interactions must be present to express the circadian molecular profiles observed in wild-type plants. A set of those essential interactions is called herein a kernel of the circadian system. The kernel structure unbiasedly reveals four interlocked negative feedback loops contributing to circadian rhythms, and three feedback loops among them drive the autonomous oscillation itself. Strikingly, the kernel structure, as well as the whole clock circuitry, is overwhelmingly composed of inhibitory, rather than activating, interactions between genes. We found that this tendency underlies plant circadian molecular profiles which often exhibit sharply-shaped, cuspidate waveforms. Through the generation of these cuspidate profiles, inhibitory interactions may facilitate the global coordination of temporally-distant clock events that are markedly peaked at very specific times of day. Our systematic approach resulting in experimentally-testable predictions provides insights into a design principle of biological clockwork, with implications for synthetic biology.     

Identification of a Gene Involved in the Control of the Root System Development in Arabidopsis thaliana

Genetic and molecular genetic analysis of a lethal root mutant of Arabidopsis thaliana was carried out. The mutant was obtained from a collection created earlier by means of insertion mutagenesis. The mutation was found to be recessive. It was caused by an insertion of the T region of vector pLD3 used for transformation of germinating seeds when creating the collection of insertion mutants. A 118-bp DNA fragment flanking the left border of the insertion was isolated using the TAIL PCR technique, and its nucleotide sequence was determined. Computer analysis of this DNA region demonstrated that it was located in exon 32 of the YUP8H12R.44 gene in chromosome 1.     

Quantitative Genetic Analysis of Thermal Dissipation in Arabidopsis

Feedback deexcitation is a photosynthetic regulatory mechanism that can protect plants from high light stress by harmlessly dissipating excess absorbed light energy as heat. To understand the genetic basis for intraspecies differences in thermal dissipation capacity, we investigated natural variation in Arabidopsis (Arabidopsis thaliana). We determined the variation in the amount of thermal dissipation by measuring nonphotochemical quenching (NPQ) of chlorophyll fluorescence in Arabidopsis accessions of diverse origins. Ll-1 and Sf-2 were selected as high NPQ Arabidopsis accessions, and Columbia-0 (Col-0) and Wassilewskija-2 were selected as relatively low NPQ accessions. In spite of significant differences in NPQ, previously identified NPQ factors were indistinguishable between the high and the low NPQ accessions. Intermediate levels of NPQ in Ll-1 × Col-0 F1 and Sf-2 × Col-0 F1 compared to NPQ levels in their parental lines and continuous distribution of NPQ in F2 indicated that the variation in NPQ is under the control of multiple nuclear factors. To identify genetic factors responsible for the NPQ variation, we developed a polymorphic molecular marker set for Sf-2 × Col-0 at approximately 10-centimorgan intervals. From quantitative trait locus (QTL) mapping with undistorted genotype data and NPQ measurements in an F2 mapping population, we identified two high NPQ QTLs, HQE1 (high qE 1, for high energy-dependent quenching 1) and HQE2, on chromosomes 1 and 2, and the phenotype of HQE2 was validated by analysis of near isogenic lines. Neither QTL maps to a gene that had been identified previously in extensive forward genetics screens using induced mutants, suggesting that quantitative genetics can be used to find new genes affecting thermal dissipation.Plants require light energy, by definition, to drive photosynthesis. However, too much light causes photooxidative damage in plants (Barber and Andersson, 1992). Thus, plants have diverse defense mechanisms against high light stress (Niyogi, 1999). For example, chloroplasts can move to absorb less light energy (Kasahara et al., 2002), and light-harvesting antenna size in chloroplasts can be reduced (Anderson, 1986). Plants also can harmlessly dissipate excess absorbed light energy as heat (Müller et al., 2001), and they have alternative electron transport pathways to relieve overreduction of electron transport components under stress conditions (Niyogi, 2000; Ort and Baker, 2002).Thermal dissipation is mediated by a mechanism called feedback deexcitation. Feedback deexcitation dissipates excess absorbed light energy as heat, thereby protecting plants from high-light stress (Horton et al., 1994; Niyogi, 1999). The amount of feedback deexcitation can be quantified by measuring nonphotochemical quenching (NPQ) of chlorophyll fluorescence (Müller et al., 2001; Baker, 2008). NPQ is induced by appearance of high light and is relaxed following disappearance of the high light. Based on its relaxation kinetics, NPQ can be divided into at least three components: energy-dependent quenching (qE), state-transition quenching, and photoinhibitory quenching (Maxwell and Johnson, 2000; Müller et al., 2001). Among them, qE is generally the major component in plants (Maxwell and Johnson, 2000). Biochemical and molecular genetics studies have shown that a pH gradient across the thylakoid membrane (Briantais et al., 1979; Munekage et al., 2001, 2002), the xanthophyll cycle (Demmig-Adams et al., 1990; Niyogi et al., 1998), and the PsbS protein (Li et al., 2000) of PSII are important factors involved in controlling the induction and/or extent of NPQ. Based on the semidominance of loss-of-function mutations (Li et al., 2000, 2002a) and overexpression of the psbS gene (Li et al., 2002b) in Arabidopsis (Arabidopsis thaliana), the expression level of the PsbS protein has been suggested as an important factor in determining the qE (and total NPQ) capacity of plants.Naturally occurring variation in NPQ capacity has been observed in different plant species (Johnson et al., 1993; Demmig-Adams and Adams, 1994; Demmig-Adams, 1998). The variation of saturated NPQ values ranges from 2.5 to 4.5 in British plant species, and plants grown in open habitats tend to have larger NPQ capacity (Johnson et al., 1993). Sun-acclimated plants contain up to four times as much NPQ capacity as low-light-acclimated plants of the same species (Osmond et al., 1993; Ruban et al., 1993; Brugnoli et al., 1994; Demmig-Adams and Adams, 1994; Demmig-Adams et al., 1995; Demmig-Adams, 1998; Roberts et al., 1998). In Monstera deliciosa, for example, sun-acclimated leaves showed higher NPQ than low-light-acclimated leaves (Demmig-Adams and Adams, 1994), and this difference in NPQ is correlated with changes in PsbS protein levels (Demmig-Adams et al., 2006). Although it has been suggested that there may be a genetic basis for the variation (Horton et al., 1994), this possibility has not yet been analyzed.This kind of natural variation in plant traits, in most cases, shows continuous variations that are under the control of polygenic factors, and quantitative genetic studies are required to understand the genetic basis of the variation (Alonso-Blanco and Koornneef, 2000). Arabidopsis has become a model system for plant quantitative trait locus (QTL) mapping (Alonso-Blanco and Koornneef, 2000; Maloof, 2003; Tonsor et al., 2005) because it has considerable trait variations among accessions, advanced molecular biological tools for efficient genotyping with molecular markers, and a fully sequenced genome. In addition, development and improvement of statistical tools for QTL mapping facilitate analyses of quantitative traits (Lander and Botstein, 1989; Zeng, 1994; Sen and Churchill, 2001). Using Arabidopsis, a number of quantitative traits have been analyzed; however, QTL mapping is still underused for photosynthesis-related traits, in spite of advantages for quantitative genetic studies, such as simple ways for quantification of photosynthetic parameters (Krause and Weis, 1991; Laisk et al., 2002; Long and Bernacchi, 2003; Baker, 2008).In this article, we report natural variation of NPQ among Arabidopsis accessions and test the hypothesis that the variation between a high NPQ accession (Sf-2) and a low NPQ accession (Columbia-0 [Col-0]) is related to the PsbS protein. We measured induction and relaxation of NPQ in Arabidopsis accessions and divided them into high and low NPQ accessions. Biochemical and molecular biological experiments did not associate the NPQ differences with PsbS or other previously identified NPQ factors. Genetic analyses revealed that the differences are controlled by polygenic nuclear factors. To identify these factors, we performed QTL mapping using Sf-2 × Col-0 F2 progeny as a mapping population and identified two high NPQ QTLs. The significance of NPQ variation and possible roles for these QTLs in thermal dissipation are discussed.     

An Efficient Method for Quantitative,Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.     

The Crosstalk between Cytokinin and Auxin Signaling Pathways in the Control of Natural Senescence of Arabidopsis thaliana Leaves

Russian Journal of Plant Physiology - Natural senescence of leaves is a regular physiological process subjected to a certain genetic program and controlled by various phytohormones. Cytokinins...     

A Simple Hydroponic Culture Method for the Development of a Highly Viable Root System in Arabidopsis thaliana

In the studies of nutritional absorption and metal toxicity in the root, it is important to grow plants without technical damage. We established a simple hydroponic culture system for Arabidopsis thaliana to obtain a healthy plant having a well-developed root system with many lateral roots. The phytotoxic effects of Cr, Cu, and Al ions were examined by FDA-PI staining using this culture system. The pattern of root inhibition varied with the ion, suggesting the usefulness of this culture system.