Plastic and genetic responses of a common sedge to warming have contrasting effects on carbon cycle processes.

Climate warming affects plant physiology through genetic adaptation and phenotypic plasticity, but little is known about how these mechanisms influence ecosystem processes. We used three elevation gradients and a reciprocal transplant experiment to show that temperature causes genetic change in the sedge Eriophorum vaginatum. We demonstrate that plants originating from warmer climate produce fewer secondary compounds, grow faster and accelerate carbon dioxide (CO2 ) release to the atmosphere. However, warmer climate also caused plasticity in E. vaginatum, inhibiting nitrogen metabolism, photosynthesis and growth and slowing CO2 release into the atmosphere. Genetic differentiation and plasticity in E. vaginatum thus had opposing effects on CO2 fluxes, suggesting that warming over many generations may buffer, or reverse, the short-term influence of this species over carbon cycle processes. Our findings demonstrate the capacity for plant evolution to impact ecosystem processes, and reveal a further mechanism through which plants will shape ecosystem responses to climate change.

Novel Micro-Phenotyping Approach to Chemical Genetic Screening for Increased Plant Tolerance to Abiotic Stress.

Studying the effects of small molecules on root system development in the context of a large-scale chemical genetic screen has previously been a technical challenge. The recent development of novel seedling growth devices ("Phytostrips"), used in combination with standard 96-well microtiter plates, has made it possible to perform detailed studies of changes in root morphology and root system architecture following the application of a library of chemical compounds. Phytostrips were originally designed to allow automated robotic capture of images of roots and shoots of the model species Arabidopsis thaliana, but can also be used for manual screens that are more laborious but do not require the investment in expensive robotics.Here we describe a protocol for the use of Phytostrips to perform chemical genetic screens that rely on clearly observable changes in root morphology or root system architecture. As an example, we describe the use of polyethylene glycol to impose an abiotic stress related to reduced water potential and the application of a chemical screen for small molecules that are able to rescue Arabidopsis root development from the disruptive effect of the polyethylene glycol treatment. The protocol we describe provides a template for the application of a multiplicity of other screens for compounds that can antagonize the effects of a range of abiotic stresses on root development.

Stomatal and growth responses to hydraulic and chemical changes induced by progressive soil drying.

A better understanding of physiological responses of crops to drought stress is important for ensuring sustained crop productivity under climate change. Here, we studied the effect on 15-day-old maize (Zea mays L.) plants of a 6 d non-lethal period of soil drying [soil water potential (SWP) decreased from -0.20 MPa to -0.81 MPa]. Root growth was initially stimulated during drying (when SWP decreased from -0.31 MPa to -0.38 MPa, compared with -0.29 MPa in well-watered pots), followed by inhibition during Days 5-6 (SWP from -0.63 MPa to -0.81 MPa). Abscisic acid (ABA) in the root began to accumulate as the root water potential declined during Days 2-3. Leaf elongation was inhibited from Day 4 (SWP less than -0.51 MPa), just after leaf ABA content began to increase, but coinciding with a decline in leaf water potential. The stomatal conductance was restricted earlier in the younger leaf (fourth) (on Day 3) than in the older leaf (third). The ethylene content of leaves and roots decreased during drying, but after the respective increase in ABA contents. This work identified critical timing of hydraulic and chemical changes at the onset of soil drying, which can be important in initiating early stomatal and growth responses to drought.

The Biphasic Root Growth Response to Abscisic Acid in Arabidopsis Involves Interaction with Ethylene and Auxin Signalling Pathways.

Exogenous abscisic acid (ABA) is known to either stimulate or inhibit root growth, depending on its concentration. In this study, the roles of ethylene and auxin in this biphasic effect of ABA on root elongation were investigated using chemical inhibitors and mutants. Inhibitors of ethylene perception and biosynthesis and an auxin influx inhibitor were all found to block the inhibitory effect of high ABA concentrations, but not the stimulatory effect of low ABA concentrations. In addition, three ethylene-insensitive mutants (etr1-1, ein2-1, and ein3-1), two auxin influx mutants (aux1-7, aux1-T) and an auxin-insensitive mutant (iaa7/axr2-1) were all insensitive to the inhibitory effect of high ABA concentrations. In the case of the stimulatory effect of low ABA concentrations, it was blocked by two different auxin efflux inhibitors and was less pronounced in an auxin efflux mutant (pin2/eir1-1) and in the iaa7/axr2-1 auxin-insensitive mutant. Thus it appears that the stimulatory effect seen at low ABA concentrations is via an ethylene-independent pathway requiring auxin signalling and auxin efflux through PIN2/EIR1, while the inhibitory effect at high ABA concentrations is via an ethylene-dependent pathway requiring auxin signalling and auxin influx through AUX1.

QTL analysis of the developmental response to L-glutamate in Arabidopsis roots and its genotype-by-environment interactions.

Primary root growth in Arabidopsis and a number of other species has previously been shown to be remarkably sensitive to the presence of external glutamate, with glutamate signalling eliciting major changes in root architecture. Using two recombinant inbred lines from reciprocal crosses between Arabidopsis accessions C24 and Col-0, we have identified one large-effect quantitative trait locus (QTL), GluS1, and two minor QTLs, GluS2 and GluS3, which together accounted for 41% of the phenotypic variance in glutamate sensitivity. The presence of the GluS1 locus on chromosome 3 was confirmed using a set of C24/Col-0 isogenic lines. GluS1 was mapped to an interval between genes At3g44830-At3g46880. When QTL mapping was repeated under a range of environmental conditions, including temperature, shading and nitrate supply, a strong genotype-by-environment interaction in the controls for the glutamate response was identified. Major differences in the loci controlling this trait were found under different environmental conditions. Here we present evidence for the existence of loci on chromosomes 1 and 5 epistatically controlling the response of the GluS1 locus to variations in ambient temperature, between 20°C and 26°C. In addition, a locus on the long arm of chromosome 1 was found to play a major role in controlling the ability of external nitrate signals to antagonize the glutamate effect. We conclude that there are multiple loci controlling natural variation in glutamate sensitivity in Arabidopsis roots and that epistatic interactions play an important role in modulating glutamate sensitivity in response to changes in environmental conditions.

The Microphenotron: a robotic miniaturized plant phenotyping platform with diverse applications in chemical biology.

BACKGROUND: Chemical genetics provides a powerful alternative to conventional genetics for understanding gene function. However, its application to plants has been limited by the lack of a technology that allows detailed phenotyping of whole-seedling development in the context of a high-throughput chemical screen. We have therefore sought to develop an automated micro-phenotyping platform that would allow both root and shoot development to be monitored under conditions where the phenotypic effects of large numbers of small molecules can be assessed. RESULTS: The 'Microphenotron' platform uses 96-well microtitre plates to deliver chemical treatments to seedlings of Arabidopsis thaliana L. and is based around four components: (a) the 'Phytostrip', a novel seedling growth device that enables chemical treatments to be combined with the automated capture of images of developing roots and shoots; (b) an illuminated robotic platform that uses a commercially available robotic manipulator to capture images of developing shoots and roots; (c) software to control the sequence of robotic movements and integrate these with the image capture process; (d) purpose-made image analysis software for automated extraction of quantitative phenotypic data. Imaging of each plate (representing 80 separate assays) takes 4 min and can easily be performed daily for time-course studies. As currently configured, the Microphenotron has a capacity of 54 microtitre plates in a growth room footprint of 2.1 m2, giving a potential throughput of up to 4320 chemical treatments in a typical 10 days experiment. The Microphenotron has been validated by using it to screen a collection of 800 natural compounds for qualitative effects on root development and to perform a quantitative analysis of the effects of a range of concentrations of nitrate and ammonium on seedling development. CONCLUSIONS: The Microphenotron is an automated screening platform that for the first time is able to combine large numbers of individual chemical treatments with a detailed analysis of whole-seedling development, and particularly root system development. The Microphenotron should provide a powerful new tool for chemical genetics and for wider chemical biology applications, including the development of natural and synthetic chemical products for improved agricultural sustainability.

AutoRoot: open-source software employing a novel image analysis approach to support fully-automated plant phenotyping.

BACKGROUND: Computer-based phenotyping of plants has risen in importance in recent years. Whilst much software has been written to aid phenotyping using image analysis, to date the vast majority has been only semi-automatic. However, such interaction is not desirable in high throughput approaches. Here, we present a system designed to analyse plant images in a completely automated manner, allowing genuine high throughput measurement of root traits. To do this we introduce a new set of proxy traits. RESULTS: We test the system on a new, automated image capture system, the Microphenotron, which is able to image many 1000s of roots/h. A simple experiment is presented, treating the plants with differing chemical conditions to produce different phenotypes. The automated imaging setup and the new software tool was used to measure proxy traits in each well. A correlation matrix was calculated across automated and manual measures, as a validation. Some particular proxy measures are very highly correlated with the manual measures (e.g. proxy length to manual length, r2 > 0.9). This suggests that while the automated measures are not directly equivalent to classic manual measures, they can be used to indicate phenotypic differences (hence the term, proxy). In addition, the raw discriminative power of the new proxy traits was examined. Principal component analysis was calculated across all proxy measures over two phenotypically-different groups of plants. Many of the proxy traits can be used to separate the data in the two conditions. CONCLUSION: The new proxy traits proposed tend to correlate well with equivalent manual measures, where these exist. Additionally, the new measures display strong discriminative power. It is suggested that for particular phenotypic differences, different traits will be relevant, and not all will have meaningful manual equivalent measures. However, approaches such as PCA can be used to interrogate the resulting data to identify differences between datasets. Select images can then be carefully manually inspected if the nature of the precise differences is required. We suggest such flexible measurement approaches are necessary for fully automated, high throughput systems such as the Microphenotron.

How do plants sense their nitrogen status?

The primary processes that contribute to the efficient capture of soil nitrate are the development of a root system that effectively explores the soil and the expression of high-affinity nitrate uptake systems in those roots. Both these processes are highly regulated to take into account the availability and distribution of external nitrate pools and the endogenous N status of the plant. While significant progress has been made in elucidating the early steps in sensing and responding to external nitrate, there is much less clarity about how the plant monitors its N status. This review specifically addresses the questions of what N compounds are sensed and in which part of the plant, as well as the identity of the signalling pathways responsible for their detection. Candidates that are considered for the role of N sensory systems include the target of rapamycin (TOR) signalling pathway, the general control non-derepressible 2 (GCN2) pathway, the plastidic PII-dependent pathway, and the family of glutamate-like receptors (GLRs). However, despite significant recent progress in elucidating the function and mode of action of these signalling systems, there is still much uncertainty about the extent to which they contribute to the process by which plants monitor their N status. The possibility is discussed that the large GLR family of Ca2+ channels, which are gated by a wide range of different amino acids and expressed throughout the plant, could act as amino acid sensors upstream of a Ca2+-regulated signalling pathway, such as the TOR pathway, to regulate the plant's response to changes in N status.

MADS-box transcription factor OsMADS25 regulates root development through affection of nitrate accumulation in rice.

MADS-box transcription factors are vital regulators participating in plant growth and development process and the functions of most of them are still unknown. ANR1 was reported to play a key role in controlling lateral root development through nitrate signal in Arabidopsis. OsMADS25 is one of five ANR1-like genes in Oryza Sativa and belongs to the ANR1 clade. Here we have investigated the role of OsMADS25 in the plant's responses to external nitrate in Oryza Sativa. Our results showed that OsMADS25 protein was found in the nucleus as well as in the cytoplasm. Over-expression of OsMADS25 significantly promoted lateral and primary root growth as well as shoot growth in a nitrate-dependent manner in Arabidopsis. OsMADS25 overexpression in transgenic rice resulted in significantly increased primary root length, lateral root number, lateral root length and shoot fresh weight in the presence of nitrate. Down-regulation of OsMADS25 in transgenic rice exhibited significantly reduced shoot and root growth in the presence of nitrate. Furthermore, over-expression of OsMADS25 in transgenic rice promoted nitrate accumulation and significantly increased the expressions of nitrate transporter genes at high rates of nitrate supply while down-regulation of OsMADS25 produced the opposite effect. Taken together, our findings suggest that OsMADS25 is a positive regulator control lateral and primary root development in rice.

Glutamate receptor-like channels in plants: a role as amino acid sensors in plant defence?

Plant glutamate receptor-like genes (GLRs) are homologous to the genes for mammalian ionotropic glutamate receptors (iGluRs), after which they were named, but in the 16 years since their existence was first revealed, progress in elucidating their biological role has been disappointingly slow. Recently, however, studies from a number of laboratories focusing on the model plant species Arabidopsis thaliana (L.) have thrown new light on the functional properties of some members of the GLR gene family. One important finding has been that plant GLR receptors have a much broader ligand specificity than their mammalian iGluR counterparts, with evidence that some individual GLR receptors can be gated by as many as seven amino acids. These results, together with the ubiquity of their expression throughout the plant, open up the possibility that GLR receptors could have a pervasive role in plants as non-specific amino acid sensors in diverse biological processes. Addressing what one of these roles could be, recent studies examining the wound response and disease susceptibility in GLR knockout mutants have provided evidence that some members of clade 3 of the GLR gene family encode important components of the plant's defence response. Ways in which this family of amino acid receptors might contribute to the plant's ability to respond to an attack from pests and pathogens are discussed.

Nitrogen signalling pathways shaping root system architecture: an update.

Root system architecture is a fundamentally important trait for resource acquisition in both ecological and agronomic contexts. Because of the plasticity of root development and the almost infinite complexity of the soil, root system architecture is shaped by environmental factors to a much greater degree than shoot architecture. In attempting to understand how roots sense and respond to environmental cues, the striking effects of nitrate and other forms of nitrogen on root growth and branching have received particular attention. This minireview focuses on the latest advances in our understanding of the diverse nitrogen signalling pathways that are now known to act at multiple stages in the process of lateral root development, as well as on primary root growth.

Glutamate signalling in roots.

As a signalling molecule, glutamate is best known for its role as a fast excitatory neurotransmitter in the mammalian nervous system, a role that requires the activity of a family of ionotropic glutamate receptors (iGluRs). The unexpected discovery in 1998 that Arabidopsis thaliana L. possesses a family of iGluR-related (GLR) genes laid the foundations for an assessment of glutamate's potential role as a signalling molecule in plants that is still in progress. Recent advances in elucidating the function of Arabidopsis GLR receptors has revealed similarities with iGluRs in their channel properties, but marked differences in their ligand specificities. The ability of plant GLR receptors to act as amino-acid-gated Ca(2+) channels with a broad agonist profile, combined with their expression throughout the plant, makes them strong candidates for a multiplicity of amino acid signalling roles. Although root growth is inhibited in the presence of a number of amino acids, only glutamate elicits a specific sequence of changes in growth, root tip morphology, and root branching. The recent finding that the MEKK1 gene is a positive regulator of glutamate sensitivity at the root tip has provided genetic evidence for the existence in plants of a glutamate signalling pathway analogous to those found in animals. This short review will discuss the most recent advances in understanding glutamate signalling in roots, considering them in the context of previous work in plants and animals.

Glutamate signalling via a MEKK1 kinase-dependent pathway induces changes in Arabidopsis root architecture.

A chemical genetic approach has been used to investigate the mechanism by which external glutamate (l-Glu) is able to trigger major changes in root architecture in Arabidopsis thaliana L. An initial screen of 80 agonists and antagonists of mammalian glutamate and GABA receptors, using a specially developed 96-well microphenotyping system, found none that replicated the response of the root to l-Glu or antagonized it. However, a larger screen using >1500 molecules bioactive in Saccharomyces cerevisiae (yeast) identified two groups that interfered with the l-Glu response. One of the antagonists, 2-(4-chloro-3-methylphenyl)-2-oxoethyl thiocyanate (CMOT), has been reported to target Ste11, an evolutionarily conserved MAP kinase kinase kinase (MAP3K) in yeast. This led to the discovery that root growth in a triple mekk1 mekk2 mekk3 mutant (mekk1/2/3), defective in a set of three tandemly arranged MAP3Ks, was almost insensitive to l-Glu. However, the sensitivity of mekk1/2/3 roots to inhibition by other amino acids reported to act as agonists of glutamate receptor-like (GLR) channels in Arabidopsis roots (Asn, Cys, Gly and Ser) was unaffected. The l-Glu sensitivity of the mekk1/2/3 mutant was restored by transformation with a construct carrying the intact MEKK1 gene. These results demonstrate that MEKK1 plays a key role in transducing the l-Glu signal that elicits large-scale changes in root architecture, and provide genetic evidence for the existence in plants of an l-Glu signalling pathway analogous to that found in animals.

Overexpressing the ANR1 MADS-box gene in transgenic plants provides new insights into its role in the nitrate regulation of root development.

The expression of the ANR1 MADS-box gene was manipulated in transgenic plants to investigate its role in the NO(3)(-)-dependent regulation of root development in Arabidopsis thaliana. Constitutive overexpression of ANR1 in roots, achieved using GAL4 enhancer trap lines, resulted in more rapid early seedling development, increased lengths and numbers of lateral roots and increased shoot fresh weight. Based on results obtained with five different enhancer trap lines, the overexpression of ANR1 in the lateral root tips appears to be more important for this phenotype than its level of expression in the developing lateral root primordia. Dexamethasone-mediated induction of ANR1 in lines expressing an ANR1-GR (glucocorticoid receptor) fusion protein stimulated lateral root growth but not primary root growth. Short-term (24 h) dexamethasone treatments led to prolonged stimulation of lateral root growth, whether the lateral roots were already mature or still unemerged at the time of treatment. In split-root experiments, localized application of dexamethasone to half of the root system of an ANR1-GR line elicited a localized increase in both the length and numbers of lateral roots, mimicking the effect of a localized NO(3)(-) treatment. In both types of transgenic line, the root phenotype was strongly dependent on the presence of NO(3)(-), indicating that there are additional components involved in ANR1 function that are NO(3)(-) regulated. The implications of these results for our understanding of ANR1's mode of action in the root response to localized NO(3)(-) are discussed.

Quantitative analysis of lateral root development: pitfalls and how to avoid them.

The advent of the postgenomics era has led to increased interest in exploring the role of gene networks and signaling pathways in controlling plant development. The last two decades have seen a particular increase in the number of studies focusing on the development of the Arabidopsis thaliana root system. However, the investigation of such a seemingly simple system as an Arabidopsis root can lead to problems in quantification and errors in interpretation if knowledge of root organization is lacking. In this article, we identify a number of these problems and give examples of potentially erroneous and correct determinations of lateral root parameters. Our aim is to bring this important issue to the attention of the plant science community and to suggest ways in which the problems inherent in quantifying the process of lateral root development can be avoided.

Is it good noise? The role of developmental instability in the shaping of a root system.

Root architecture plays a major part in determining a root system's ability to function effectively and efficiently in its essential roles of anchorage and the capture of soil resources. The characteristics of root development that are conventionally considered to be the main determinants of root architecture are the rate, angle, and duration of root growth and the pattern of root branching. In this review, the case is made that there is an additional trait that has been largely ignored but which has a significant influence on root architecture, namely the degree to which stochasticity (or 'developmental instability') affects the developmental process. Although the intrinsic variability in the development and growth of lateral roots has been recognized for some time, in almost every study of root development this remarkable facet of root behaviour tends to be hidden beneath the veil of statistical averaging. Progress in other fields is providing intriguing insights into the phenomenon of developmental instability, how it is generated at the molecular and cellular levels and the genetic mechanisms by which it is buffered. This review will consider the existence of developmental instability in roots, its underlying causes, its effects on root architecture, and the evidence that it is under genetic control. The hypothesis will be advanced that developmental instability in roots is an adaptive trait, and its potential relevance to root function will be discussed in both an ecological and an agronomic context.

Nitrate and glutamate as environmental cues for behavioural responses in plant roots.

As roots explore the soil, they encounter a complex and fluctuating environment in which the different edaphic resources (water and nutrients) are heterogeneously distributed in space and time. Many plant species are able to respond to this heterogeneity by modifying their root system development, such that they colonize the most resource-rich patches of soil. The complexities of these responses, and their dependence on the implied ability to perceive and integrate multiple external signals, would seem to amply justify the term 'behaviour'. This review will consider the types of behaviour that are elicited in roots of Arabidopsis thaliana by exposure to variations in the external concentrations and distribution of two different N compounds, nitrate and glutamate. Molecular genetic studies have revealed an intricate N regulatory network at the root tip that is responsible for orchestrating changes in root growth rate and root architecture to accommodate variations in the extrinsic and intrinsic supply of N. The review will discuss what is known of the genetic basis for these responses and speculate on their physiological and ecological significance.

Nitrate signalling mediated by the NRT1.1 nitrate transporter antagonises L-glutamate-induced changes in root architecture.

Arabidopsis root architecture is highly responsive to changes in the nitrogen supply. External NO(3)(-) stimulates lateral root growth via a signalling pathway involving the ANR1 MADS box transcription factor, while the presence of exogenous l-glutamate (Glu) at the primary root tip slows primary root growth and stimulates root branching. We have found that NO(3)(-), in conjunction with Glu, has a hitherto unrecognized role in regulating the growth of primary roots. Nitrate was able to stimulate primary root growth, both directly and by antagonising the inhibitory effect of Glu. Each response depended on direct contact between the primary root tip and the NO(3)(-), and was not elicited by an alternative N source (NH(4)(+)). The chl1-5 mutant, which is defective in the NRT1.1 (CHL1) NO(3)(-) transporter, was insensitive to NO(3)(-) antagonism of Glu signalling, while an anr1 mutant retained its sensitivity. Sensitivity to NO(3)(-) was restored in a chl1-5 mutant constitutively expressing NRT1.1. However, expression in chl1-5 of a transport-competent but non-phosphorylatable form of NRT1.1 not only failed to restore NO(3)(-) sensitivity but also had a dominant-negative effect on Glu sensitivity. Our results indicate the existence of a NO(3)(-) signalling pathway at the primary root tip that can antagonise the root's response to Glu, and they further suggest that NRT1.1 has a direct NO(3)(-) sensing role in this pathway. We discuss how the observed signalling interactions between NO(3)(-) and Glu could provide a mechanism for modulating root architecture in response to changes in the relative abundance of organic and inorganic N.