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.

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.

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.

L-Glutamate as a Novel Modifier of Root Growth and Branching: What's the Sensor?

Exogenous L-glutamate (L-Glu) has been shown to be able to elicit major changes in Arabidopsis root architecture at micromolar concentrations. The root response, which is strongly genotype-dependent, is specific to L-Glu and involves both inhibition of primary root growth and stimulation of root branching behind the primary root tip. The L-Glu appears to be sensed directly at the root tip, where it inhibits meristematic activity. An intriguing and still unanswered question is whether members of the family of Glu receptor-like genes (GLRs) have a role in mediating this response. A pharmacological approach described here, using agonists and antagonists of mammalian ionotropic Glu receptors, has failed to resolve the issue. Progress towards identifying the genes involved in the root response to L-Glu is likely to come through the application of forward and reverse genetics, in combination with quantitative trait loci (QTL) mapping.

Evidence that L-glutamate can act as an exogenous signal to modulate root growth and branching in Arabidopsis thaliana.

The roots of many plant species are known to use inorganic nitrogen, in the form of , as a cue to initiate localized root proliferation within nutrient-rich patches of soil. We report here that, at micromolar concentrations and in a genotype-dependent manner, exogenous l-glutamate is also able to elicit complex changes in Arabidopsis root development. l-Glutamate is perceived specifically at the primary root tip and inhibits mitotic activity in the root apical meristem, but does not interfere with lateral root initiation or outgrowth. Only some time after emergence do lateral roots acquire l-glutamate sensitivity, indicating that their ability to respond to l-glutamate is developmentally regulated. Comparisons between different Arabidopsis ecotypes revealed a remarkable degree of natural variation in l-glutamate sensitivity, with C24 being the most sensitive. The aux1-7 auxin transport mutant had reduced l-glutamate sensitivity, suggesting a possible interaction between l-glutamate and auxin signaling. Surprisingly, two loss-of-function mutants at the AXR1 locus (axr1-3 and axr1-12) were hypersensitive to l-glutamate. A pharmacological approach, using agonists and antagonists of mammalian ionotropic glutamate receptors, was unable to provide evidence of a role for their plant homologs in sensing exogenous glutamate. We discuss the mechanism of l-glutamate sensing and the possible ecological significance of the observed l-glutamate-elicited changes in root architecture.

Nitrogen regulation of root branching.

BACKGROUND: Many plant species can modify their root architecture to enable them to forage for heterogeneously distributed nutrients in the soil. The foraging response normally involves increased proliferation of lateral roots within nutrient-rich soil patches, but much remains to be understood about the signalling mechanisms that enable roots to sense variations in the external concentrations of different mineral nutrients and to modify their patterns of growth and development accordingly. SCOPE: In this review we consider different aspects of the way in which the nitrogen supply can modify root branching, focusing on Arabidopsis thaliana. Our current understanding of the mechanism of nitrate stimulation of lateral root growth and the role of the ANR1 gene are summarized. In addition, evidence supporting the possible role of auxin in regulating the systemic inhibition of early lateral root development by high rates of nitrate supply is presented. Finally, we examine recent evidence that an amino acid, L-glutamate, can act as an external signal to elicit complex changes in root growth and development. CONCLUSIONS: It is clear that plants have evolved sophisticated pathways for sensing and responding to changes in different components of the external nitrogen supply as well as their own internal nitrogen status. We speculate on the possibility that the effects elicited by external L-glutamate represent a novel form of foraging response that could potentially enhance a plant's ability to compete with its neighbours and micro-organisms for localized sources of organic nitrogen.

Signaling mechanisms integrating root and shoot responses to changes in the nitrogen supply.

During their life cycle, plants must be able to adapt to wide variations in the supply of soil nitrogen (N). Changes in N availability, and in the relative concentrations of NO(3) (-)and NH(4) (+), are known to have profound regulatory effects on the N uptake systems in the root, on C and N metabolism throughout the plant, and on root and shoot morphology. Optimising the plant's responses to fluctuations in the N supply requires co-ordination of the pathways of C and N assimilation, as well as establishment of the appropriate allocation of resources between root and shoot growth. Achieving this integration of responses at the whole plant level implies long-distance signaling mechanisms that can communicate information about the current availability of N from root-to-shoot, and information about the C/N status of the shoot in the reverse direction. In this review we will discuss recent advances which have contributed to our understanding of these long-range signaling pathways.

Root-derived cytokinins as long-distance signals for NO3--induced stimulation of leaf growth.

Leaf growth of many plant species shows rapid changes in response to alterations of the form and the level of N supply. In hydroponically-grown tomato (Lycopersicon esculentum L.), leaf growth was rapidly stimulated by NO(3)(-) application to NH(4)(+) precultured plants, while NH(4)(+) supply or complete N deprivation to NO(3)(-) precultured plants resulted in a rapid inhibition of leaf growth. Just 10 microM NO(3)(-) supply was sufficient to stimulate leaf growth to the same extent as 2 mM. Furthermore, continuous NO(3)(-) supply induced an oscillation of leaf growth rate with a 48 h interval. Since changes in NO(3)(-) levels in the xylem exudate and leaves did not correlate with NO(3)(-)-induced alterations of leaf growth rate, additional signals such as phytohormones may be involved. Levels of a known inhibitor of leaf growth, abscisic acid (ABA), did not consistently correspond to leaf growth rates in wild-type plants. Moreover, leaf growth of the ABA-deficient tomato mutant flacca was inhibited by NH(4)(+) without an increase in ABA concentration and was stimulated by NO(3)(-) despite its excessive ethylene production. These findings suggest that neither ABA nor ethylene are directly involved in the effects of N form on leaf growth. However, under all experimental conditions, stimulation of leaf growth by NO(3)(-) was consistently associated with increased concentration of the physiologically active forms of cytokinins, zeatin and zeatin riboside, in the xylem exudate. This indicates a major role for cytokinins as long-distance signals mediating the shoot response to NO(3)(-) perception in roots.

Elevated pCO(2 )favours nitrate reduction in the roots of wild-type tobacco (Nicotiana tabacum cv. Gat.) and significantly alters N-metabolism in transformants lacking functional nitrate reductase in the roots.

The impact of elevated pCO(2 )on N-metabolism of hydroponically grown wild-type and transformed tobacco plants lacking root nitrate reduction was studied in order to elucidate the effects on (i) nitrate uptake, (ii) long-distance transport of N, (iii) nitrate reduction with emphasis on root-NR, and (iv) the allocation of N between the root and shoot. The findings were related to alterations of growth rates. At elevated pCO(2 )the wild type exhibited higher growth rates, which were accompanied by an increase of NO(3)(-)-uptake per plant, due to a higher root:shoot ratio. Furthermore, elevated pCO(2 )enhanced nitrate reduction in the roots of the wild type, resulting in enhanced xylem-loading of organic N (amino-N) to supply the shoot with sufficient nitrogen, and decreased phloem-transport of organic N in a basipetal direction. Transformed tobacco plants lacking root nitrate reduction were smaller than the wild type and exhibited lower growth rates. Nitrate uptake per plant was decreased in transformed plants as a consequence of an impeded root growth and, thus, a significantly decreased root:shoot ratio. Surprisingly, transformed plants showed an altered allocation of amino-N between the root and the shoot, with an increase of amino-N in the root and a substantial decrease of amino-N in the shoot. In transformed plants, xylem-loading of nitrate was increased and the roots were supplied with organic N via phloem transport. Elevated pCO(2 )increased shoot-NR, but only slightly affected the growth rates of transformed plants, whereas carbohydrates accumulated at elevated pCO(2 )as indicated by a significant increase of the C/N ratio in the leaves of transformed plants. Unexpectedly, the C/N balance and the functional equilibrium between root and shoot growth was disturbed dramatically by the loss of nitrate reduction in the root.