Growth kinetics, spatialization and quality of potato tubers monitored in situ by MRI - long-term effects of water stress.

Understanding the potato tuber development and effects of drought at key stages of sensitivity on yield is crucial, particularly when considering the increasing incidence of drought due to climate change. So far, few studies addressed the time course of tuber growth in soil, mainly due to difficulties in accessing underground plant organs in a non-destructive manner. This study aims to understand the tuber growth and quality and the complex long-term effects of realistic water stress on potato tuber yield. MRI was used to monitor the growth kinetics and spatialization of individual tubers in situ and the evolution of internal defects throughout the development period. The intermittent drought applied to plants reduced tuber yield by reducing tuber growth and increasing the number of aborted tubers. The reduction in the size of tubers depended on the vertical position of the tubers in the soil, indicating water exchanges between tubers and the mother plant during leaf dehydration events. The final size of tubers was linked with the growth rate at specific developmental periods. For plants experiencing stress, this corresponded to the days following rewatering, suggesting tuber growth plasticity. All internal defects occurred in large tubers and within a short time span immediately following a period of rapid growth of perimedullary tissues, probably due to high nutrient requirements. To conclude, the non-destructive 3D imaging by MRI allowed us to quantify and better understand the kinetics and spatialization of tuber growth and the appearance of internal defects under different soil water conditions.

Brassica napus Drought-Induced 22-kD Protein (BnD22) Acts Simultaneously as a Cysteine Protease Inhibitor and Chlorophyll-Binding Protein.

Class II water-soluble chlorophyll proteins (WSCPs) from Brassicaceae are non-photosynthetic proteins that bind with chlorophyll (Chl) and its derivatives. The physiological function of WSCPs is still unclear, but it is assumed to be involved in stress responses, which is likely related to their Chl-binding and protease inhibition (PI) activities. Yet, the dual function and simultaneous functionality of WSCPs must still be better understood. Here, the biochemical functions of Brassica napus drought-induced 22-kDa protein (BnD22), a major WSCP expressed in B. napus leaves, were investigated using recombinant hexahistidine-tagged protein. We showed that BnD22 inhibited cysteine proteases, such as papain, but not serine proteases. BnD22 was able to bind with Chla or Chlb to form tetrameric complexes. Unexpectedly, BnD22-Chl tetramer displays higher inhibition toward cysteine proteases, indicating (i) simultaneous Chl-binding and PI activities and (ii) Chl-dependent activation of PI activity of BnD22. Moreover, the photostability of BnD22-Chl tetramer was reduced upon binding with the protease. Using three-dimensional structural modeling and molecular docking, we revealed that Chl binding favors interaction between BnD22 and proteases. Despite its Chl-binding ability, the BnD22 was not detected in chloroplasts but rather in the endoplasmic reticulum and vacuole. In addition, the C-terminal extension peptide of BnD22, which cleaved off post-translationally in vivo, was not implicated in subcellular localization. Instead, it drastically promoted the expression, solubility and stability of the recombinant protein.

Molecular evolution and transcriptional regulation of the oilseed rape proline dehydrogenase genes suggest distinct roles of proline catabolism during development.

MAIN CONCLUSION: Six BnaProDH1 and two BnaProDH2 genes were identified in Brassica napus genome. The BnaProDH1 genes are mainly expressed in pollen and roots' organs while BnaProDH2 gene expression is associated with leaf vascular tissues at senescence. Proline dehydrogenase (ProDH) catalyzes the first step in the catabolism of proline. The ProDH gene family in oilseed rape (Brassica napus) was characterized and compared to other Brassicaceae ProDH sequences to establish the phylogenetic relationships between genes. Six BnaProDH1 genes and two BnaProDH2 genes were identified in the B. napus genome. Expression of the three paralogous pairs of BnaProDH1 genes and the two homoeologous BnaProDH2 genes was measured by real-time quantitative RT-PCR in plants at vegetative and reproductive stages. The BnaProDH2 genes are specifically expressed in vasculature in an age-dependent manner, while BnaProDH1 genes are strongly expressed in pollen grains and roots. Compared to the abundant expression of BnaProDH1, the overall expression of BnaProDH2 is low except in roots and senescent leaves. The BnaProDH1 paralogs showed different levels of expression with BnaA&C.ProDH1.a the most strongly expressed and BnaA&C.ProDH1.c the least. The promoters of two BnaProDH1 and two BnaProDH2 genes were fused with uidA reporter gene (GUS) to characterize organ and tissue expression profiles in transformed B. napus plants. The transformants with promoters from different genes showed contrasting patterns of GUS activity, which corresponded to the spatial expression of their respective transcripts. ProDHs probably have non-redundant functions in different organs and at different phenological stages. In terms of molecular evolution, all BnaProDH sequences appear to have undergone strong purifying selection and some copies are becoming subfunctionalized. This detailed description of oilseed rape ProDH genes provides new elements to investigate the function of proline metabolism in plant development.

A profiling approach of the natural variability of foliar N remobilization at the rosette stage gives clues to understand the limiting processes involved in the low N use efficiency of winter oilseed rape.

Oilseed rape, a crop requiring a high level of nitogen (N) fertilizers, is characterized by low N use efficiency. To identify the limiting factors involved in the N use efficiency of winter oilseed rape, the response to low N supply was investigated at the vegetative stage in 10 genotypes by using long-term pulse-chase (15)N labelling and studying the physiological processes of leaf N remobilization. Analysis of growth and components of N use efficiency allowed four profiles to be defined. Group 1 was characterized by an efficient N remobilization under low and high N conditions but by a decrease of leaf growth under N limitation. Group 2 showed a decrease in leaf growth under low N supply that was associated with a low N remobilization efficiency under both N supplies despite a high remobilization of soluble proteins. In response to N limitation, Group 3 is characterized by an increase in N use efficiency and leaf N remobilization compared with high N that is not sufficient to sustain the leaf biomass production at a similar level to non-limited plants. Genotypes of Group 4 subjected to low nitrate were able to maintain leaf growth to the same level as under high N. The profiling approach indicated that enhancement of amino acid export and soluble protein degradation was crucial for N remobilization improvement. At the whole-plant level, N fluxes revealed that Group 4 showed a high N remobilization in source leaves combined with a better N utilization in young leaves. Consequently, an enhanced N remobilization limits N loss in fallen leaves, but this remobilized N needs to be efficiently utilized in young leaves to improve N use efficiency.

γ-Aminobutyric acid transaminase deficiency impairs central carbon metabolism and leads to cell wall defects during salt stress in Arabidopsis roots.

Environmental constraints challenge cell homeostasis and thus require a tight regulation of metabolic activity. We have previously reported that the γ-aminobutyric acid (GABA) metabolism is crucial for Arabidopsis salt tolerance as revealed by the NaCl hypersensitivity of the GABA transaminase (GABA-T, At3g22200) gaba-t/pop2-1 mutant. In this study, we demonstrate that GABA-T deficiency during salt stress causes root and hypocotyl developmental defects and alterations of cell wall composition. A comparative genome-wide transcriptional analysis revealed that expression levels of genes involved in carbon metabolism, particularly sucrose and starch catabolism, were found to increase upon the loss of GABA-T function under salt stress conditions. Consistent with the altered mutant cell wall composition, a number of cell wall-related genes were also found differentially expressed. A targeted quantitative analysis of primary metabolites revealed that glutamate (GABA precursor) accumulated while succinate (the final product of GABA metabolism) significantly decreased in mutant roots after 1 d of NaCl treatment. Furthermore, sugar concentration was twofold reduced in gaba-t/pop2-1 mutant roots compared with wild type. Together, our results provide strong evidence that GABA metabolism is a major route for succinate production in roots and identify GABA as a major player of central carbon adjustment during salt stress.

Modulation of ethylene biosynthesis by ACC and AIB reveals a structural and functional relationship between the K15NO3 uptake rate and root absorbing surfaces.

The modification of root traits in relation to nitrate uptake represents a source for improvement of nitrogen uptake efficiency. Because ethylene signalling modulates growth of exploratory and root hair systems more rapidly (minutes to hours) than nitrate signalling (days to weeks), a pharmacological approach was used to decipher the relationships between root elongation and N uptake. Rape seedlings were grown on agar plates supplied with 1mM K(15)NO3 and treated with different concentrations of either the ethylene precursor, ACC (0.1, 1, and 10 µM) or an inhibitor of ethylene biosynthesis, AIB (0.5 and 1 µM). The results showed that rapid modulation of root elongation (up to 8-fold) is more dependent on the ethylene than the nitrate signal. Indeed, ACC treatment induced a partial compensatory increase in (15)N uptake associated with overexpression of the BnNRT2.1 and BnNRT1.1 genes. Likewise, daily root elongation between treatments was not associated with daily nitrate uptake but was correlated with N status. This suggested that a part of the daily root response was modulated by cross talks between ethylene signalling and N and C metabolisms. This was confirmed by the reduction in C allocation to the roots induced by ACC treatment and the correlations of changes in the root length and shoot surface area with the aspartate content. The observed effects of ethylene signalling in the root elongation and NRT gene expression are discussed in the context of the putative role of NRT2.1 and NRT1.1 transporters as nitrate sensors.

Arginase induction represses gall development during clubroot infection in Arabidopsis.

Arginase induction can play a defensive role through the reduction of arginine availability for phytophageous insects. Arginase activity is also induced during gall growth caused by Plasmodiophora brassicae infection in roots of Arabidopsis thaliana; however, its possible role in this context has been unclear. We report here that the mutation of the arginase-encoding gene ARGAH2 abrogates clubroot-induced arginase activity and results in enhanced gall size in infected roots, suggesting that arginase plays a defensive role. Induction of arginase activity in infected roots was impaired in the jar1 mutant, highlighting a link between the arginase response to clubroot and jasmonate signaling. Clubroot-induced accumulation of the principal amino acids in galls was not affected by the argah2 mutation. Because ARGAH2 was previously reported to control auxin response, we investigated the role of ARGAH2 in callus induction. ARGAH2 was found to be highly induced in auxin/cytokinin-triggered aseptic plant calli, and callus development was enhanced in argah2 in the absence of the pathogen. We hypothesized that arginase contributes to a negative control over clubroot symptoms, by reducing hormone-triggered cellular proliferation.

New insights into chlorophyll-WSCP (water-soluble chlorophyll proteins) interactions : The case study of BnD22 (Brassica napus drought-induced 22 kDa).

The water-soluble chlorophyll-proteins (WSCP) of class II from Brassicaceae are non-photosynthetic proteins that bind chlorophylls (Chls) and chlorophyll derivatives. Their physiological roles, biochemical functions and mode of action are still unclear. It is assumed that the WSCPs have a protection function against Chl photodamage during stressful conditions. WSCPs are subdivided into class IIA and class IIB according to their apparent Chla/b binding ratio. Although their Chla/Chlb binding selectivity has been partly characterized, their Chl affinities are not yet precisely defined. For instance, WSCPs IIA do not show any Chl binding preference while WSCPs IIB have greater affinity to Chlb. In this study, we present a novel method for assessment of Chl binding to WSCPs based on the differences of Chl photobleaching rates in a large range of Chl/protein ratios. The protein we have chosen to study WSCP is BnD22, a WSCP IIA induced in the leaves of Brassica napus under water deficit. BnD22 formed oligomeric complexes upon binding to Chla and/or Chlb allowing a protective effect against photodamage. The binding constants indicate that BnD22 binds with high affinity the Chls and with a strong selectivity to Chla. Moreover, dependending of Chl/protein ratio upon reconstitution, two distinct binding events were detected resulting from difference of Chl stoichiometry inside oligomeric complexes.

Characterization of the Water Shortage Effects on Potato Tuber Tissues during Growth Using MRI Relaxometry and Biochemical Parameters.

The potato is one of the most cultivated crops worldwide, providing an important source of food. The quality of potato tubers relates to their size and dry matter composition and to the absence of physiological defects. It depends on the spatial and temporal coordination of growth and metabolic processes in the major tuber tissues: the cortex, flesh and pith. In the present study, variations in the biochemical traits of each of these tissues were investigated during tuber growth under optimal and water-deficit conditions. MRI relaxometry was used as a non-invasive and quantitative method to access information on cellular water status. The presence of slight but significant variations in organic compound contents quantified in the cortex and flesh revealed a tissue-dependent metabolic pattern. The T2 and relative I0 of the bi-exponential relaxation signal allowed a distinction to be made between the pith and the cortex, whereas the flesh could be differentiated from these tissues only through its relative I0. T2 values did not vary significantly during tuber development, in accordance with the typical growth pattern of tubers, but were shown to be sensitive to water stress. The interpretation of the multi-exponential transverse relaxation times is discussed and could be further developed via microscopic analysis.

GABA accumulation causes cell elongation defects and a decrease in expression of genes encoding secreted and cell wall-related proteins in Arabidopsis thaliana.

GABA (γ-aminobutyric acid), a non-protein amino acid, is a signaling factor in many organisms. In plants, GABA is known to accumulate under a variety of stresses. However, the consequence of GABA accumulation, especially in vegetative tissues, remains poorly understood. Moreover, gene expression changes as a consequence of GABA accumulation in plants are largely unknown. The pop2 mutant, which is defective in GABA catabolism and accumulates GABA, is a good model to examine the effects of GABA accumulation on plant development. Here, we show that the pop2 mutants have pollen tube elongation defects in the transmitting tract of pistils. Additionally, we observed growth inhibition of primary root and dark-grown hypocotyl, at least in part due to cell elongation defects, upon exposure to exogenous GABA. Microarray analysis of pop2-1 seedlings grown in GABA-supplemented medium revealed that 60% of genes whose expression decreased encode secreted proteins. Besides, functional classification of genes with decreased expression in the pop2-1 mutant showed that cell wall-related genes were significantly enriched in the microarray data set, consistent with the cell elongation defects observed in pop2 mutants. Our study identifies cell elongation defects caused by GABA accumulation in both reproductive and vegetative tissues. Additionally, our results show that genes that encode secreted and cell wall-related proteins may mediate some of the effects of GABA accumulation. The potential function of GABA as a growth control factor under stressful conditions is discussed.

Genetic and physiological analysis of the relationship between partial resistance to clubroot and tolerance to trehalose in Arabidopsis thaliana.

In Arabidopsis thaliana the induction of plant trehalase during clubroot disease was proposed to act as a defense mechanism in the susceptible accession Col-0, which could thereby cope with the accumulation of pathogen-synthesized trehalose. In the present study, we assessed trehalose activity and tolerance to trehalose in the clubroot partially resistant accession Bur-0. We compared both accessions for several trehalose-related physiological traits during clubroot infection. A quantitative trait loci (QTLs) analysis of tolerance to exogenous trehalose was also conducted on a Bur-0xCol-0 RIL progeny. Trehalase activity was not induced by clubroot in Bur-0 and the inhibition of trehalase by validamycin treatments resulted in the enhancement of clubroot symptoms only in Col-0. In pathogen-free cultures, Bur-0 showed less trehalose-induced toxicity symptoms than Col-0. A QTL analysis identified one locus involved in tolerance to trehalose overlapping the confidence interval of a QTL for resistance to Plasmodiophora brassicae. This colocalization was confirmed using heterogeneous inbred family (HIF) lines. Although not based on trehalose catabolism capacity, partial resistance to clubroot is to some extent related to the tolerance to trehalose accumulation in Bur-0. These findings support an original model where contrasting primary metabolism-related regulations could contribute to the partial resistance to a plant pathogen.

Heterogenous Impact of Water Warming on Exotic and Native Submerged and Emergent Plants in Outdoor Mesocosms.

Some aquatic plants present high biomass production with serious consequences on ecosystem functioning. Such mass development can be favored by environmental factors. Temperature increases are expected to modify individual species responses that could shape future communities. We explored the impact of rising water temperature on the growth, phenology, and metabolism of six macrophytes belonging to two biogeographic origins (exotic, native) and two growth forms (submerged, emergent). From June to October, they were exposed to ambient temperatures and a 3 °C warming in outdoor mesocosms. Percent cover and canopy height were favored by warmer water for the exotic emergent Ludwigia hexapetala. Warming did not modify total final biomass for any of the species but led to a decrease in total soluble sugars for all, possibly indicating changes in carbon allocation. Three emergent species presented lower flavonol and anthocyanin contents under increased temperatures, suggesting lower investment in defense mechanisms and mitigation of the stress generated by autumn temperatures. Finally, the 3 °C warming extended and shortened flowering period for L. hexapetala and Myosotis scorpioides, respectively. The changes generated by increased temperature in outdoor conditions were heterogenous and varied depending on species but not on species biogeographic origin or growth form. Results suggest that climate warming could favor the invasiveness of L. hexapetala and impact the structure and composition of aquatic plants communities.

The Arabidopsis pop2-1 mutant reveals the involvement of GABA transaminase in salt stress tolerance.

BACKGROUND: GABA (gamma-aminobutyric acid) is a non protein amino acid that has been reported to accumulate in a number of plant species when subjected to high salinity and many other environmental constraints. However, no experimental data are to date available on the molecular function of GABA and the involvement of its metabolism in salt stress tolerance in higher plants. Here, we investigated the regulation of GABA metabolism in Arabidopsis thaliana at the metabolite, enzymatic activity and gene transcription levels upon NaCl stress. RESULTS: We identified the GABA transaminase (GABA-T), the first step of GABA catabolism, as the most responsive to NaCl. We further performed a functional analysis of the corresponding gene POP2 and demonstrated that the previously isolated loss-of-function pop2-1 mutant was oversensitive to ionic stress but not to osmotic stress suggesting a specific role in salt tolerance. NaCl oversensitivity was not associated with overaccumulation of Na+ and Cl- but mutant showed a slight decrease in K+. To bring insights into POP2 function, a promoter-reporter gene strategy was used and showed that POP2 was mainly expressed in roots under control conditions and was induced in primary root apex and aerial parts of plants in response to NaCl. Additionally, GC-MS- and UPLC-based metabolite profiling revealed major changes in roots of pop2-1 mutant upon NaCl stress including accumulation of amino acids and decrease in carbohydrates content. CONCLUSIONS: GABA metabolism was overall up-regulated in response to NaCl in Arabidopsis. Particularly, GABA-T was found to play a pivotal function and impairment of this step was responsible for a decrease in salt tolerance indicating that GABA catabolism was a determinant of Arabidopsis salt tolerance. GABA-T would act in salt responses in linking N and C metabolisms in roots.

Combined Allosteric Responses Explain the Bifurcation in Non-Linear Dynamics of 15N Root Fluxes Under Nutritional Steady-State Conditions for Nitrate.

With regard to thermodynamics out of equilibrium, seedlings are open systems that dissipate energy towards their environment. Accordingly, under nutritional steady-state conditions, changes in external concentrations of one single ion provokes instability and reorganization in the metabolic and structure/architecture of the seedling that is more favorable to the fluxes of energy and matter. This reorganization is called a bifurcation and is described in mathematics as a non-linear dynamic system. In this study, we investigate the non-linear dynamics of 15N fluxes among cellular compartments of B. napus seedlings in response to a wide range of external NO 3 - 15 concentrations (from 0.05 to 20 mM): this allows to determine whether any stationary states and bifurcations could be found. The biphasic behavior of the root NO 3 - 15 uptake rate (vin ) was explained by the combined cooperative properties between the vapp (N uptake, storage and assimilation rate) and vout (N translocation rate) 15N fluxes that revealed a unique and stable stationary state around 0.28 mM nitrate. The disappearance of this stationary state around 0.5 mM external nitrate concentrations provokes a dramatic bifurcation in 15N flux pattern. This bifurcation in the vin and vout 15N fluxes fits better with the increase of BnNPF6.3/NRT1.1 expression than BnNRT2.1 nitrate transporter genes, confirming the allosteric property of the BnNPF6/NRT1.1 transporter, as reported in the literature between low and high nitrate concentrations. Moreover, several statistically significant power-law equations were found between variations in the shoots tryptophan concentrations (i.e., IAA precursor) with changes in the vapp and vout 15N fluxes as well as a synthetic parameter of plant N status estimated from the root/shoot ratio of total free amino acids concentrations. These relationships designate IAA as one of the major biological parameters related to metabolic and structural-morphological reorganization coupled with the N and water fluxes induced by nitrate. The results seriously challenge the scientific grounds of the concept of high- and low-affinity of nitrate transporters and are therefore discussed in terms of the ecological significance and physiological implications on the basis of recent agronomic, physiological and molecular data of the literature.

Inhibition of Aminotransferases by Aminoethoxyvinylglycine Triggers a Nitrogen Limitation Condition and Deregulation of Histidine Homeostasis That Impact Root and Shoot Development and Nitrate Uptake.

Background and Aims: Although AVG (aminoethoxyvinylglycine) is intensely used to decipher signaling in ethylene/indol-3-acetic acid (IAA) interactions on root morphogenesis, AVG is not a specific inhibitor of aminocyclopropane-1-carboxylate synthase (ACS) and tryptophan aminotransferase (TAA) and tryptophan aminotransferase related (TAR) activities since it is able to inhibit several aminotransferases involved in N metabolism. Indeed, 1 mM glutamate (Glu) supply to the roots in plants treated with 10 µM AVG partially restores the root growth. Here, we highlight the changes induced by AVG and AVG + Glu treatments on the N metabolism impairment and root morphogenetic program. Methods: Root nitrate uptake induced by AVG and AVG + Glu treatments was measured by a differential labeling with 15NO3 - and 15Nglutamate. In parallel a profiling of amino acids (AA) was performed to decipher the impairment of AA metabolism. Key Results: 10 µM AVG treatment increases K15NO3 uptake and 15N translocation during root growth inhibition whereas 10 µM AVG + 1 mM 15Nglutamate treatment inhibits K15NO3 uptake and increases 15Nglutamate uptake during partial root growth restoration. This is explained by a nitrogen (N) limitation condition induced by AVG treatment and a N excess condition induced by AVG + Glu treatment. AA levels were mainly impaired by AVG treatment in roots, where levels of Ser, Thr, α-Ala, ß-Ala, Val, Asn and His were significantly increased. His was the only amino acid for which no restoration was observed in roots and shoots after glutamate treatment suggesting important control of His homeostasis on aminotransferase network. Results were discussed in light of recent findings on the interconnection between His homeostasis and the general amino acid control system (GAAC) in eukaryotes. Conclusions: These results demonstrate that AVG concentration above 5 µM is a powerful pharmacological tool for unraveling the involvement of GAAC system or new N sensory system in morphological and metabolic changes of the roots in leguminous and non-leguminous plants.

Germination and Seedling Growth of Water Primroses: A Cross Experiment between Two Invaded Ranges with Contrasting Climates.

Aquatic ecosystems are vulnerable to biological invasions, and will also be strongly impacted by climate change, including temperature increase. Understanding the colonization dynamics of aquatic invasive plant species is of high importance for preservation of native biodiversity. Many aquatic invasive plants rely on clonal reproduction to spread, but mixed reproductive modes are common. Under future climate changes, these species may favor a sexual reproductive mode. The aim of this study was to test the germination capacity and the seedling growth of two water primrose species, Ludwigia hexapetala and Ludwigia peploides, both invasive in Europe and in the United States. We performed a reciprocal transplant of seeds of L. hexapetala and L. peploides from two invasive ranges into experimental gardens characterized by Oceanic and Mediterranean-type climates. Our results showed that higher temperatures increased or maintained germination percentages and velocity, decreased survivorship of germinants, but increased their production of biomass. The origin of the seeds had low impact on L. hexapetala responses to temperature, but greatly influenced those of L. peploides. The invasiveness of water primroses in ranges with Oceanic climates might increase with temperature. The recruitment from seed banks by these species should be considered by managers to improve the conservation of native aquatic and wetland plant species.

Characterization of the salt stress vulnerability of three invasive freshwater plant species using a metabolic profiling approach.

The effects of salt stress on freshwater plants has been little studied up to now, despite the fact that they are expected to present different levels of salt sensitivity or salt resistance depending on the species. The aim of this work was to assess the effect of NaCl at two concentrations on three invasive freshwater species, Elodea canadensis, Myriophyllum aquaticum and Ludwigia grandiflora, by examining morphological and physiological parameters and using metabolic profiling. The growth rate (biomass and stem length) was reduced for all species, whatever the salt treatment, but the response to salt differed between the three species, depending on the NaCl concentration. For E. canadensis, the physiological traits and metabolic profiles were only slightly modified in response to salt, whereas M. aquaticum and L. grandiflora showed great changes. In both of these species, root number, photosynthetic pigment content, amino acids and carbohydrate metabolism were affected by the salt treatments. Moreover, we are the first to report the salt-induced accumulation of compatible solutes in both species. Indeed, in response to NaCl, L. grandiflora mainly accumulated sucrose. The response of M. aquaticum was more complex, because it accumulated not only sucrose and myo-inositol whatever the level of salt stress, but also amino acids such as proline and GABA, but only at high NaCl concentrations. These responses are the metabolic responses typically found in terrestrial plants.

Effects of the inhibitor of the γ-aminobutyrate-transaminase, vinyl-γ-aminobutyrate, on development and nitrogen metabolism in Brassica napus seedlings.

γ-aminobutyrate-transaminase (EC 2.6.1.19) catalyzes the first step of the catabolism of γ-aminobutyric acid (GABA), a non-protein amino acid well-known to accumulate in plant in response to environmental stimuli. Recent studies reinforce more and more the role of its metabolism in carbon and/or nitrogen metabolisms and as a signalling molecule in developmental processes. Here we investigated the effects of inhibition of γ-aminobutyrate-transaminase (GABA-T) in seedlings of Brassica napus, using vinyl-GABA (VGB) as a specific inhibitor of GABA-T to prevent enzyme activity. Root growth was reduced by 44% in VGB-treated seedlings but was less inhibited when VGB was associated with exogenous GABA and was not reduced with exogenous GABA alone. Measurements of GABA content in seedlings grown on VGB, GABA or VGB + GABA demonstrated that GABA level in root was not linked with the root length reduction, suggesting that GABA was not the sole component acting in root growth inhibition. Besides, metabolic profiling revealed that in root, VGB-treatment caused a twofold increase in content of almost all amino acids, except for alanine whose content was 19-fold higher than in control. In order to test the involvement of alanine accumulation on growth we studied the effects of exogenous alanine. High alanine content slightly reduced root growth suggesting that VGB-induced alanine accumulation was not responsible for root length reduction. We conclude that root growth inhibition in plants whose GABA catabolism was impaired could result at least partly from the disruption of the primary metabolism as a whole rather than direct effect of GABA on cellular growth process.

In low transpiring conditions, uncoupling the BnNrt2.1 and BnNrt1.1 NO 3(-) transporters by glutamate treatment reveals the essential role of BnNRT2.1 for nitrate uptake and the nitrate-signaling cascade during growth.

In plants, the nitrate transporters, NRT1.1 and NRT2.1, are mainly responsible for nitrate uptake. Intriguingly, both nitrate transporters are located in a complementary manner in different cells layers of the mature root suggesting that their coordination should occur during nitrate uptake and plant growth. This hypothesis was examined on 5-d-old rape seedlings grown on agar medium supplemented with 1 or 5mM nitrate. Seedlings were treated with increasing potassium glutamate concentrations in order to uncouple the two nitrate transporters by inhibiting BnNRT2.1 expression and activity specifically. In both nitrate treatments, increasing the glutamate concentrations from 0.5 to 10mM induced a reduction in (15)NO 3(-) uptake and an inhibition of N assimilation. The decrease in (15)NO 3(-) uptake was caused by downregulation of BnNRT2.1 expression but surprisingly it was not compensated by the upregulation of BnNRT1.1. This created an unprecedented physiological situation where the effects of the nitrate signal on shoot growth were solely modulated by nitrate absorption. In these conditions, the osmotic water flow for volumetric shoot growth was mainly dependent on active nitrate transport and nitrate signaling. This behavior was confirmed by the allometric relationships found between changes in the root length with (15)N and water accumulation in the shoot. These findings demonstrate that the BnNRT2.1 transporter is essential for nitrate uptake and growth, and renew the question of the respective roles of the NRT2.1 and NRT1.1 transporters in nitrate uptake and sensing at the whole plant level.

In low transpiring conditions, nitrate and water fluxes for growth of B. napus plantlets correlate with changes in BnNrt2.1 and BnNrt1.1 transporter expression.

We analyzed how changes in BnNrt nitrate transporter gene expression induced by nitrate are associated with morphological changes in plantlets and osmotic water flow for growth. We hypothesized that in a Petri dish system, reduction in transpiration should induce conditions where nitrate and water fluxes for growth depend directly on nitrate transporter activity and nitrate signaling. Rape seedlings growing on agar plates were supplied with increasing external K (15)NO 3 concentrations from 0.05 to 20 mM. After 5 d of treatment, morphological switches in plantlet growth were observed between 0.5 and 5 mM nitrate supply. Root elongation was reduced by 50% while the cotyledon surface area was doubled. These morphological switches were strongly associated with increases in (15)NO 3(-) and water uptake rates as well as (15)N and water allocation to the shoot. These switches were also highly correlated with the upregulation of BnNrt1.1 and BnNrt2.1 in the root. However, while root expression of BnNrt2.1 was correlated linearly with a shoot growth-associated increase in (15)N and water uptake, BnNrt1.1 expression was correlated exponentially with both (15)N and water accumulation. In low transpiring conditions, the tight control exercised by nitrate transporters on K (15)NO 3 uptake and allocation clearly demonstrates that they modulated the nitrate-signaling cascade involved in cell growth and as a consequence, water uptake and allocation to the growing organs. Deciphering this signaling cascade in relation to acid growth theory seems to be the most important challenge for our understanding of the nitrate-signaling role in plant growth.