Nitrogen (N) is probably the most important macronutrient and its scarcity limits plant growth, development and fitness. N starvation response has been largely studied by transcriptomic analyses, but little is known about the role of alternative polyadenylation (APA) in such response. In this work, we show that N starvation modifies poly(A) usage in a large number of transcripts, some of them mediated by FIP1, a component of the polyadenylation machinery. Interestingly, the number of mRNAs isoforms with poly(A) tags located in protein-coding regions or 5 '-UTRs significantly increases in response to N starvation. The set of genes affected by APA in response to N deficiency is enriched in N-metabolism, oxidation-reduction processes, response to stresses, and hormone responses, among others. A hormone profile analysis shows that the levels of salicylic acid (SA), a phytohormone that reduces nitrate accumulation and root growth, increase significantly upon N starvation. Meta-analyses of APA-affected and fip1-2-deregulated genes indicate a connection between the nitrogen starvation response and salicylic acid (SA) signaling. Genetic analyses show that SA may be important for preventing the overgrowth of the root system in low N environments. This work provides new insights on how plants interconnect different pathways, such as defense-related hormonal signaling and the regulation of genomic information by APA, to fine-tune the response to low N availability.

The jasmonate signaling pathway regulates development, growth, and defense responses in plants. Studies in the model eudicot, Arabidopsis thaliana, have identified the bioactive hormone (jasmonoylisoleucine [JA-Ile]) and its Coronatine Insensitive 1 (COI1)/Jasmonate-ZIM Domain (JAZ) co-receptor. In bryophytes, a conserved signaling pathway regulates similar responses but uses a different ligand, the JA-Ile precursor dinor-12-oxo-10,15(Z)-phytodienoic acid (dn-OPDA), to activate a conserved co-receptor. Jasmonate responses independent of JA-Ile and COI1, thought to be mediated by the cyclopentenone OPDA, have also been suggested, but experimental limitations in Arabidopsis have hindered attempts to uncouple OPDA and JA-Ile biosynthesis. Thus, a clear understanding of this pathway remains elusive. Here, we address the role of cyclopentenones in COI1-independent responses using the bryophyte Marchantia polymorpha, which is unable to synthesize JA-Ile but does accumulate OPDA and dn-OPDA. We demonstrate that OPDA and dn-OPDA activate a COI1-independent pathway that regulates plant thermotolerance genes, and consequently, treatment with these oxylipins protects plants against heat stress. Furthermore, we identify that these molecules signal through their electrophilic properties.

The jasmonate (JA)-pathway regulators MYC2, MYC3, and MYC4 are central nodes in plant signaling networks integrating environmental and developmental signals to fine-tune JA defenses and plant growth. Continuous activation of MYC activity is potentially lethal. Hence, MYCs need to be tightly regulated in order to optimize plant fitness. Among the increasing number of mechanisms regulating MYC activity, protein stability is arising as a major player. However, how the levels of MYC proteins are modulated is still poorly understood. Here, we report that MYC2, MYC3, and MYC4 are targets of BPM (BTB/POZ-MATH) proteins, which act as substrate adaptors of CUL3-based E3 ubiquitin ligases. Reduction of function of CUL3(BPM) in amiR-bpm lines, bpm235 triple mutants, and cul3ab double mutants enhances MYC2 and MYC3 stability and accumulation and potentiates plant responses to JA such as root-growth inhibition and MYC-regulated gene expression. Moreover, MYC3 polyubiquitination levels are reduced in amiR-bpm lines. BPM3 protein is stabilized by JA, suggesting a negative feedback regulatory mechanism to control MYC activity, avoiding harmful runaway responses. Our results uncover a layer for JA-pathway regulation by CUL3(BPM)-mediated degradation of MYC transcription factors.

The global decrease in soil fertility leads to a new agricultural scenario where eco-friendly solutions play an important role. The plant growth promotion through the use of microbes, especially endophytes and rhizosphere microbiota, has been proposed as a useful solution. Several studies have shown that humic substances are suitable vehicles for the inoculation of plant growth promoting bacteria, and that this combination has an enhanced effect on the stimulation of plant development. In this work, cucumber plants grown hydroponically have been pre-treated with a sedimentary humic acid (SHA) with known plant growth-enhancing effects, and culturable bacterial endophytes have been isolated from these plants. The hypothesis was that this pre-treatment with SHA could lead to the isolation of certain endophytic taxa whose proliferation within the plant could have been promoted as a result of the effects of the treatment with SHA, and that could eventually reinforce a potential synergistic effect of a combined application of those endophytic bacteria and SHA. The culturable endophytes that have been isolated from humic acid-treated cucumber plants have been identified as members of four main phyla:Proteobacteria,Firmicutes,Actinobacteria, andBacteroidetes. Isolates were characterized according to the following plant growth-promoting traits: nitrogen fixation/scavenging, phosphate solubilization, siderophore production and plant hormone production. Most of the isolates were able to fix/scavenge nitrogen and to produce plant hormones (indole-3-acetic acid and several cytokinins), whereas few isolates were able to solubilize phosphate and/or produce siderophores. The most promising endophyte isolates for its use in futures investigations as plant growth-promoting bacterial inocula werePseudomonassp. strains (that showed all traits),Sphingomonassp.,Stenotrophomonassp. strains, or someArthrobactersp. andMicrobacteriumsp. isolates.

Humic substances (HS, fulvic and humic acids) are widely used as fertilizers or plant growth stimulants, although their mechanism of action still remains partially unknown. Humic substances may be applied either directly to the soil or as foliar sprays. Despite both kind of application are commonly used in agricultural practices, most of the studies regarding the elicited response in plants induced by HS are based on the root-application of these substances. The present work aimed at discriminating between the mechanisms of action of foliar application versus root application of a sedimentary humic acid (SHA) on plant development. For this purpose, six markers related to plant phenotype, plant morphology, hormonal balance and root-plasma membrane H+-ATPase were selected. Both application strategies improved the shoot and root growth. Foliar applied- and root applied-SHA shared the capacity to increase the concentration of indole-3-acetic acid in roots and cytokinins in shoots. However, foliar application did not lead to short-term increases in either abscisic acid root-concentration or root-plasma membrane H+-ATPase activity which are, however, two crucial effects triggered by SHA root-application. Both application modes increased the root concentrations of jasmonic acid and jasmonoyl-isoleucine. These hormonal changes caused by foliar application could be a stress-related symptom and connected to the loss of leaves trichomes and the diminution of chloroplasts size seen by scanning electron microscopy. These results support the hypothesis that the beneficial effects of SHA applied to roots or leaves may result from plant adaptation to a mild transient stress caused by SHA application.

While the general effect of CO2 enrichment on photosynthesis, stomatal conductance, N content, and yield has been documented, there is still some uncertainty as to whether there are interactive effects between CO2 enrichment and other factors, such as temperature, geographical location, water availability, and cultivar. In addition, the metabolic coordination between leaves and grains, which is crucial for crop responsiveness to elevated CO2, has never been examined closely. Here, we address these two aspects by multi-level analyses of data from several free-air CO2 enrichment experiments conducted in five different countries. There was little effect of elevated CO2 on yield (except in the USA), likely due to photosynthetic capacity acclimation, as reflected by protein profiles. In addition, there was a significant decrease in leaf amino acids (threonine) and macroelements (e.g. K) at elevated CO2, while other elements, such as Mg or S, increased. Despite the non-significant effect of CO2 enrichment on yield, grains appeared to be significantly depleted in N (as expected), but also in threonine, the S-containing amino acid methionine, and Mg. Overall, our results suggest a strong detrimental effect of CO2 enrichment on nutrient availability and remobilization from leaves to grains.

Drought stress is one of the most devastating abiotic stresses, compromising crop growth, reproductive success and yield. The arbuscular mycorrhizal (AM) symbiosis has been demonstrated to be beneficial in helping the plant to bear with water deficit. In plants, development and stress responses are largely regulated by a complex hormonal crosstalk. Auxins play significant roles in plant growth and development, in responses to different abiotic stresses or in the establishment and functioning of the AM symbiosis. Despite these important functions, the role of indole-3acetic acid (IAA) as a regulator of root water transport and stress response is not well understood. In this study, the effect of exogenous application of IAA on the regulation of root radial water transport in AM plants was analyzed under well-watered and drought stress conditions. Exogenous IAA application affected root hydraulic parameters, mainly osmotic root hydraulic conductivity (Lo), which was decreased in both AM and non-AM plants under water deficit conditions. Under drought, the relative apoplastic water flow was differentially regulated by IAA application in non-AM and AM plants. The effect of IAA on the internal cell component of root water conductivity suggests that aquaporins are involved in the IAA-dependent inhibition of this water pathway.

Silicon has not been cataloged as an essential element for higher plants. However, it has shown beneficial effects on many crops, especially under abiotic and biotic stresses. Silicon fertilization was evaluated for the first time on plants exposed to fluctuations in an Fe regime (Fe sufficiency followed by Fe deficiency and, in turn, by Fe resupply). Root and foliar Si applications were compared using cucumber plants that were hydroponically grown in a growth chamber under different Fe nutritional statuses and Si applied either to the roots or to the shoots. The SPAD index, Fe, and Mn concentration, ROS, total phenolic compounds, MDA concentration, phytohormone balance, and cell cycle were determined. The results obtained showed that the addition of Si to the roots induced an Fe shortage in plants grown under optimal or deficient Fe nutritional conditions, but this was not observed when Si was applied to the leaves. Plant recovery following Fe resupply was more effective in the Si-treated plants than in the untreated plants. A relationship between the ROS concentration, hormonal balance, and cell cycle under different Fe regimes and in the presence or absence of Si was also studied. The contribution of Si to this signaling pathway appears to be related more to the induction of Fe deficiency, than to any direct biochemical or metabolic processes. However, these roles could not be completely ruled out because several hormone differences could only be explained by the addition

JAZ proteins are negative regulators of jasmonate responses, acting both as repressors of transcription factors and as co-receptors of JA-Ile. The high redundancy of JAZ genes in angiosperms has hindered the characterization of a complete depletion of JAZ function. Moreover, the recent discovery that dn-OPDA is the jasmonate ligand in Marchantia polymorpha demonstrates that JA-Ile is not the sole COI1/JAZ ligand in land plants and highlights the importance of studying JAZ co-receptors in bryophytes. Here, we have exploited the low gene redundancy of the liverwort M. polymorpha to characterize the single MpJAZ in this early diverging plant lineage. We clarify the phylogenetic history of the TIFY family, demonstrate that MpJAZ is the ortholog of AtJAZ with a conserved function, and characterize its repressor activity of dn-OPDA responses. Our results show that, consistent with previous findings in Arabidopsis, MpJAZ represses jasmonates biosynthesis, senescence, and plant defenses, and promotes cell growth and reproductive fitness, highlighting the power of studies in Marchantia.

Evolutionary molecular plant-microbe interactions (EvoMPMI) is an emerging field bridging the gap between molecular phytopathology and evolutionary studies. EvoMPMI research is currently challenging due to the scarcity of pathogenic model systems in early-diverging land plants. Liverworts are among the earliest diverging land-plant lineages, and Marchantia polymorpha has emerged as a liverwort model for evolutionary studies. However, bacterial pathogens of Marchantia have not yet been discovered, and the molecular mechanisms controlling plant-pathogen interactions in this early-diverging land plant remain unknown. Here, we describe a robust experimental plant-bacterial pathosystem for EvoMPMI studies and discover that an ancient immune system governs plant-microbe interactions between M. polymorpha and the hemi-biotrophic pathogenic bacteria Pseudomonas syringae. We show that P. syringae pv tomato (Pto) DC3000, causal agent of tomato bacterial speck disease, colonizes M. polymorpha and activates typical hallmarks of plant innate immunity. Virulence of Pto DC3000 on M. polymorpha relies on effector activities inside liverwort cells, including conserved AvrPto and AvrPtoB functions. Host specificity analyses uncovered pathogenic differences among P. syringae strains, suggesting that M. polymorpha-P. syringae interactions are controlled by the genetic backgrounds of both host and pathogen.

The plant endosomal trafficking pathway controls the abundance of membrane-associated soluble proteins, as shown for abscisic acid (ABA) receptors of the PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS (PYR/PYL/RCAR) family. ABA receptor targeting for vacuolar degradation occurs through the late endosome route and depends on FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING1 (FYVE1) and VACUOLAR PROTEIN SORTING23A (VPS23A), components of the ENDOSOMAL SORTING COMPLEX REQUIRED FOR TRANSPORT-I (ESCRT-I) complexes. FYVE1 and VPS23A interact with ALG-2 INTERACTING PROTEIN-X (ALIX), an ESCRT-III-associated protein, although the functional relevance of such interactions and their consequences in cargo sorting are unknown. In this study we show that Arabidopsis (Arabidopsis thaliana) ALIX directly binds to ABA receptors in late endosomes, promoting their degradation. Impaired ALIX function leads to altered endosomal localization and increased accumulation of ABA receptors. In line with this activity, partial loss-of-function alix-1 mutants display ABA hypersensitivity during growth and stomatal closure, unveiling a role for the ESCRT machinery in the control of water loss through stomata. ABA-hypersensitive responses are suppressed in alix-1 plants impaired in PYR/PYL/RCAR activity, in accordance with ALIX affecting ABA responses primarily by controlling ABA receptor stability. ALIX-1 mutant protein displays reduced interaction with VPS23A and ABA receptors, providing a molecular basis for ABA hypersensitivity in alix-1 mutants. Our findings unveil a negative feedback mechanism triggered by ABA that acts via ALIX to control the accumulation of specific PYR/PYL/RCAR receptors.

The total area under quinoa (Chenopodium quinoa Willd.) cultivation and the consumption of its grain have increased in recent years because of its nutritional properties and ability to grow under adverse conditions, such as drought. Climate change scenarios predict extended periods of drought and this has emphasized the need for new crops that are tolerant to these conditions. The main goal of this work was to evaluate crop yield and quality parameters and to characterize the physiology of two varieties of quinoa grown under water deficit in greenhouse conditions. Two varieties of quinoa from the Chilean coast (Rainbow) and altiplano (Illpa) were used, grown under full irrigation or two different levels of water deficit applied during the grain filling period. There were no marked differences in yield and quality parameters between treatments, but the root biomass was higher in plants grown under severe water deficit conditions compared to control. Photosynthesis, transpiration and stomatal conductance decreased with increased water stress in both cultivars, but the coastal variety showed higher water use efficiency and less discrimination of C-13 under water deficit. This response was associated with greater root development and a better stomatal opening adjustment, especially in the case of Rainbow.

Iron (Fe) and phosphorus (P) are two essential mineral nutrients whose acquisition by plants presents important environmental and economic implications. Both elements are abundant in most soils but scarcely available to plants. To prevent Fe or P deficiency dicot plants initiate morphological and physiological responses in their roots aimed to specifically acquire these elements. The existence of common signals in Fe and P deficiency pathways suggests the signaling factors must act in conjunction with distinct nutrient-specific signals in order to confer tolerance to each deficiency. Previous works have shown the existence of cross talk between responses to Fe and P deficiency, but details of the associated signaling pathways remain unclear. Herein, the impact of foliar application of either P or Fe on P and Fe responses was studied in P- or Fe-deficient plants of Arabidopsis thaliana, including mutants exhibiting altered Fe or P homeostasis. Ferric reductase and acid phosphatase activities in roots were determined as well as the expression of genes related to P and Fe acquisition. The results obtained showed that Fe deficiency induces the expression of P acquisition genes and phosphatase activity, whereas P deficiency induces the expression of Fe acquisition genes and ferric reductase activity, although only transitorily. Importantly, these responses were reversed upon foliar application of either Fe or P on nutrient-starved plants. Taken together, the results reveal interactions between P- and Fe-related phloem signals originating in the shoots that likely interact with hormones in the roots to initiate adaptive mechanisms to tolerate deficiency of each nutrient.

Availability of fresh water for crop irrigation is becoming scarce and rather expensive. In this context, the research about the potential reutilization of non-conventional water sources becomes highly relevant, principally in arid and semi-arid areas. On many occasions, these new water resources involve water with a moderate concentration of salt, making it necessary to improve plant growth under moderate saline conditions. Besides plant breeding techniques, the use of molecules able to improve plant adaptation to saline conditions has great interest. Between these molecules, humic substances (HS) have proven to be efficient as stress-protectors under specific conditions of stress intensity and moment of application. The HS are main components of the soil organic matter and dissolved organic matter resulting from the biotic and abiotic transformation of fresh organic matter in natural ecosystems. Although knowledge about their structure is still under open debate, HS contain aromatic and aliphatic domains presenting O-, N- and S- containing functional groups with high biological and chemical activities. The aim of this presentation is to summarize the main effects of humic acids (HA) applied either on the root or on the shoot, on the metabolism and hormonal balance of plants cultivated under normal and stressing conditions, from studies carried out for our group and collaborations during the last years.

The relatively better performance of mycorrhizal plants subjected to drought stress has commonly been linked to improved root water uptake through the fungal regulation of plant aquaporins and hormones. In this study, we examined the role of ectomycorrhizal fungi in plant water relations and plant hormonal balance under mild drought using split-root seedlings of Populus trichocarpa x deltoides either with or without inoculation with Laccaria bicolor. The root compartments where the drought treatment was applied had higher ABA and lower cytokinin tZR contents, and greater expression of the plant aquaporins PtPIP1;1, PtPIP1;2, PtPIP2;5, and PtPIP2;7. On the other hand, the presence of L. bicolor within the roots down-regulated PtPIP1;4, PtPIP2;3, and PtPIP2;10, and reduced the abundance of PIP2 proteins. In addition, expression of the fungal aquaporins JQ585595 and JQ585596 were positively correlated with root ABA content, while tZR content was positively correlated with PtPIP1;4 and negatively correlated with PtPIP2;7. The results demonstrate a coordinated plant-fungal system that regulates the different mechanisms involved in water uptake in ectomycorrhizal poplar plants.

Phosphate (Pi) is an essential nutrient for all organisms. Roots are underground organs, but the majority of the root biology studies have been done on root systems growing in the presence of light. Root illumination alters the Pi starvation response (PSR) at different intensities. Thus, we have analyzed morphological, transcriptional and physiological responses to Pi starvation in dark-grown roots. We have identified new genes and pathways regulated by Pi starvation that were not described previously. We also show that Pi-starved plants increase the cis-zeatin (cZ) : trans-zeatin (tZ) ratio. Transcriptomic analyses show that tZ preferentially represses cell cycle and PSR genes, whereas cZ induces genes involved in cell and root hair elongation and differentiation. In fact, cZ-treated seedlings show longer root system as well as longer root hairs compared with tZ-treated seedlings, increasing the total absorbing surface. Mutants with low cZ concentrations do not allocate free Pi in roots during Pi starvation. We propose that Pi-starved plants increase the cZ : tZ ratio to maintain basal cytokinin responses and allocate Pi in the root system to sustain its growth. Therefore, cZ acts as a PSR hormone that stimulates root and root hair elongation to enlarge the root absorbing surface and to increase Pi concentrations in roots.

Although the ability of humic (HA) and fulvic acids (FA) to improve plant growth has been demonstrated, knowledge about the mechanisms responsible for the direct effects of HA and FA on the promotion of plant growth is scarce and fragmentary. Our study investigated the causal role of both root PM H+-ATPase activity and ABA in the SHA-promoting action on both root and shoot growth. The involvement of these processes in the regulation of shoot cytokinin concentration and activity was also studied. Our aim was to integrate such plant responses for providing new insights to the current model on the mode of action of HA for promoting root and shoot growth. Experiments employing specific inhibitors and using Cucumis sativus L. plants show that both the root PM H+-ATPase activity and root ABA play a crucial role in the root growth-promoting action of SHA. With regard to the HA-promoting effects on shoot growth, two pathways of events triggered by the interaction of SHA with plant roots are essential for the increase in root PM H+-ATPase activity-which also mediates an increase in cytokinin concentration and action in the shoot-and the ABA-mediated increase in hydraulic conductivity (Lp(r)).

Ethylene, nitric oxide (NO) and glutathione (GSH) increase in Fe-deficient roots of Strategy I species where they participate in the up-regulation of Fe acquisition genes. However, S-nitrosoglutathione (GSNO), derived from NO and GSH, decreases in Fe-deficient roots. GSNO content is regulated by the GSNO-degrading enzyme S-nitrosoglutathione reductase (GSNOR). On the other hand, there are several results showing that the regulation of Fe acquisition genes does not solely depend on hormones and signaling molecules (such as ethylene or NO), which would act as activators, but also on the internal Fe content of plants, which would act as a repressor. Moreover, different results suggest that total Fe in roots is not the repressor of Fe acquisition genes, but rather the repressor is a Fe signal that moves from shoots to roots through the phloem [hereafter named LOng Distance Iron Signal (LODIS)]. To look further in the possible interactions between LODIS, ethylene and GSNOR, we compared Arabidopsis WT Columbia and LODIS-deficient mutant opt3-2 plants subjected to different Fe treatments that alter LODIS content. The opt3-2 mutant is impaired in the loading of shoot Fe into the phloem and presents constitutive expression of Fe acquisition genes. In roots of both Columbia and opt3-2 plants we determined 1-aminocyclopropane1-carboxylic acid (ACC, ethylene precursor), expression of ethylene synthesis and signaling genes, and GSNOR expression and activity. The results obtained showed that both 'ethylene' (ACC and the expression of ethylene synthesis and signaling genes) and 'GSNOR' (expression and activity) increased in Fe-deficient WT Columbia roots. Additionally, Fe-sufficient opt3-2 roots had higher 'ethylene' and 'GSNOR' than Fe-sufficient WT Columbia roots. The increase of both 'ethylene' and 'GSNOR' was not related to the total root Fe content but to the absence of a Fe shoot signal (LODIS), and was associated with the up-regulation of Fe acquisition genes. The possible relationship between GSNOR(GSNO) and ethylene is discussed.

Biosynthesis of the phytohormone jasmonoyl-isoleucine (JA-Ile) requires reduction of the JA precursor 12-oxo-phytodienoic acid (OPDA) by OPDA reductase 3 (OPR3). Previous analyses of the opr3-1 Arabidopsis mutant suggested an OPDA signaling role independent of JA-Ile and its receptor COI1; however, this hypothesis has been challenged because opr3-1 is a conditional allele not completely impaired in JA-Ile biosynthesis. To clarify the role of OPR3 and OPDA in JA-independent defenses, we isolated and characterized a loss-of-function opr3-3 allele. Strikingly, opr3-3 plants remained resistant to necrotrophic pathogens and insect feeding, and activated COI1-dependent JA-mediated gene expression. Analysis of OPDA derivatives identified 4,5-didehydro-JA in wounded wild-type and opr3-3 plants. OPR2 was found to reduce 4,5-didehydro-JA to JA, explaining the accumulation of JA-Ile and activation of JA-Ile-responses in opr3-3 mutants. Our results demonstrate that in the absence of OPR3, OPDA enters the beta-oxidation pathway to produce 4,5-ddh-JA as a direct precursor of JA and JA-Ile, thus identifying an OPR3-independent pathway for JA biosynthesis.

Entomopathogenic fungi have traditionally been assumed to help regulate insect populations. However, some hypocrealean ascomycetes, such as Beauveria bassiana, play other, poorly understood ecological roles that might be useful in developing novel strategies for both increased crop production and crop protection. The primary aims of this work were (a) to assess endophytic colonization of bread wheat and durum wheat plants by the applied fungus B. bassiana strain EABb 04/01-Tip; (b) to examine the impact of various B. bassiana inoculation methods on growth, yield, phytohormone levels and nutrient uptake in the plants, and (c) to quantify mortality of cotton leafworm (Spodoptera littoralis) larvae fed with leaves from inoculated plants. Three experiments involving different inoculation methods (viz., 'soil treatment', 'seed dressing' and 'leaf spraying'), and a fourth experiment to assess mortality in S. littoralis larvae fed with leaves from endophytically-colonized plants, and were conducted according to a completely randomized design. Beauveria bassiana successfully established within, and colonized, bread wheat and durum wheat plants. The fungus was, for the first time, re-isolated from grains produced by plants inoculated using the 'seed dressing' and 'soil treatment' methods. The fungus boosted spike production in bread wheat inoculated using the 'seed dressing' and 'soil treatment' methods, and also in durum wheat but only using the 'soil treatment' method. 'Seed dressing' increased grain yield by about 40%, and also root length, in bread wheat compared with control plants. Mortality in S. littoralis larvae fed with leaves from inoculated plants ranged from 30% using the 'seed dressing' method to 57% using the 'leaf spraying' method compared with 0% when fed the control leaves. However, no fungal outgrowth was detected in larval cadavers. The sustainability of crop production and crop protection strategies based on B. bassiana therefore depends on the effectiveness of the inoculation method and on the particular host plant.

Climate change is leading to the intensification of drought effects worldwide, which considerably reduce crop production. A better understanding of the drought-tolerance mechanisms would lead into a more productive agriculture. The arbuscular mycorrhizal (AM) symbiosis has been shown to improve plant tolerance to drought. Salicylic acid (SA) is a phenolic compound involved in many aspects of plant growth and development. Apart from its role in biotic interactions, it is also involved in the regulation of important plant physiological processes, including plant water relations under stressful conditions. However, despite the importance of SA in plant physiology and in AM colonization, little is known about its effect on regulation of root water transport. Thus, the aim of this work was to study the combined effect of AM symbiosis and SA on root hydraulic properties under drought stress, with special focus on how these factors can alter radial root water transport pathways through aquaporin regulation. Also, the crosstalk between SA and other phytohormones was taken into account. Results showed that the AM symbiosis modifies root hydraulic responses to drought episodes. Under these conditions, AM plants showed increased Lpr and Lo. Exogenous SA application decreased Lpr and Lo under drought. SA modulation of water conductivity could be due to a fine-regulation of root aquaporins (as ZmPIP2:4 or ZmTIP1;1). Furthermore, SA application differently modulated the percentage of water flowing by the apoplastic pathway, decreasing its contribution to total root water flow in AM plants and increasing it in non-AM plants. An intricate relationship between Lpr, aquaporins and endogenous levels of SA, ABA and jasmonic acid was observed. Future studies should explore more in detail the crosstalk mechanism between these hormones in the regulation of water transport in AM roots, in order to better understand the mechanism through which the AM symbiosis copes with drought stress. (C) 2017 Elsevier B.V. All rights reserved.

Iron deficiency in plants is caused by a low availability of iron in the soil, and its main visual symptom is leaf yellowing due to a decrease in chlorophyll content, along with a reduction in plant growth and fruit quality. Foliar sprays with Fe compounds are an economic alternative to the treatment with expensive synthetic Fe-chelates applied to the soil, although the efficacy of foliar treatments is rather limited. Generally, plant response to Fe-foliar treatments is monitored by measuring chlorophyll content (or related parameters as SPAD index). However, different studies have shown that foliar Fe sprays cause a local regreening and that translocation of the applied Fe within the plant is quite low. In this context, the aim of this study was to assess the effects of foliar applications of different Fe compounds [FeSO4, Fe(III)-EDTA, and Fe(III)-heptagluconate] on Fe-deficient cucumber plants, by studying the main physiological plant root responses to Fe deficiency [root Fe(III) chelate reductase (FCR) activity; acidification of the nutrient solution; and expression of the Fe deficiency responsive genes encoding FCR, CsFRO1, Fe(II) root transporter CsIRT1, and two plasma membrane H+-ATPases, CsHA1 and CsHA2], along with SPAD index, plant growth and Fe content. The results showed that the overall assessment of Fe-deficiency root responses improved the evaluation of the efficacy of the Fe-foliar treatments compared to just monitoring SPAD indexes. Thus, FCR activity and expression of Fe-deficiency response genes, especially CsFRO1 and CsHA1, preceded the trend of SPAD index and acted as indicators of whether the plant was sensing or not metabolically active Fe due to the treatments. Principal component analysis of the data also provided a graphical tool to evaluate the evolution of plant responses to foliar Fe treatments with time.

The ability of rhizospheric humic substances to improve plant growth has been well established by many studies carried out using diverse plant species cultivated under many different conditions. These beneficial effects of humic substances on plant development are expressed in both root and shoot. However, the mechanisms responsible for this action of humic substances are only partially known and poorly integrated. In fact, although the studies focused on plant root development are numerous, those dealing with plant shoot development are scarce. Likewise, studies integrating humic effects on root and shoot are also few. In this context, the main goal of this work is to summarize some of the results regarding the effects of humic substances on plant development within a hypothetical holistic framework that will allow us to interconnect these findings and disclose some features of the functional crosstalk between the effects on soil, root and shoot. Furthermore, the significance of all these mechanisms in plants growing in the field is also discussed.

Jasmonic acid (JA) and arbuscular mycorrhizal (AM) symbioses are known to protect plants against abiotic and biotic stresses, but are also involved in the regulation of root hydraulic conductance (L). The objective of this experiment was to elucidate the role of JA in the water relations and hormonal regulation of AM plants under drought by using tomato plants defective in the synthesis of JA (def-1). Our results showed that JA is involved in the uptake and transport of water through its effect on both physiological parameters (stomatal conductance and L) and molecular parameters, mainly by controlling the expression and abundance of aquaporins. We observed that def-1 plants increased the expression of seven plant aquaporin genes under well-watered conditions in the absence of AM fungus, which partly explain the increment of L by this mutation under well-watered conditions. In addition, the effects of the AM symbiosis on plants were modified by the def-1 mutation, with the expression of some aquaporins and plant hormone concentration being disturbed. On the other hand, methyl salicylate (MeSA) content was increased in non-mycorrhizal def-1 plants, suggesting that MeSA and JA can act together in the regulation of L. In a complementary experiment, it was found that exogenous MeSA increased L, confirming our hypothesis. Likewise, we confirmed that JA, ABA and SA are hormones involved in plant mechanisms to cope with stressful situations, their concentrations being controlled by the AM symbiosis. In conclusion, under well-watered conditions, the def-1 mutation mimics the effects of AM symbiosis, but under drought conditions the def-1 mutation changed the effects of the AM symbiosis on plants.

The phytohormone jasmonoyl-isoleucine (JA-Ile) regulates defense, growth and developmental responses in vascular plants. Bryophytes have conserved sequences for all JA-Ile signaling pathway components but lack JA-Ile. We show that, in spite of 450 million years of independent evolution, the JA-Ile receptor COI1 is functionally conserved between the bryophyte Marchantia polymorpha and the eudicot Arabidopsis thaliana but COI1 responds to different ligands in each species. We identified the ligand of Marchantia MpCOI1 as two isomeric forms of the JA-Ile precursor dinor-OPDA (dinor-cis-OPDA and dinor-iso-OPDA). We demonstrate that AtCOI1 functionally complements Mpcoi1 mutation and confers JA-Ile responsiveness and that a single-residue substitution in MpCOI1 is responsible for the evolutionary switch in ligand specificity. Our results identify the ancestral bioactive jasmonate and clarify its biosynthetic pathway, demonstrate the functional conservation of its signaling pathway, and show that JA-Ile and COI1 emergence in vascular plants required co-evolution of hormone biosynthetic complexity and receptor specificity.

Arbuscular mycorrhizal symbiosis is a promising tool for improving the quality of grapes under changing environments. Therefore, the aim of this research was to determine if the ability of arbuscular mycorrhizal fungi (AMF) to enhance phenolic content (specifically, anthocyanins) in a climate change framework could be mediated by alterations in berry ABA metabolism during ripening. The study was carried out on fruit-bearing cuttings of cv. Tempranillo (CL-1048 and CL-1089) inoculated (+M) or not (¿M) with AMF. Two experimental designs were implemented. In the first experiment +M and -M plants were subjected to two temperatures (24/14¿°C or 28/18¿°C (day/night)) from fruit set to berry maturity. In the second experiment, +M and -M plants were subjected to two temperatures (24/14¿°C or 28/18¿°C (day/night)) combined with two irrigation regimes (late water deficit (LD) and full irrigation (FI)). At 28/18¿°C AMF contributed to an increase in berry anthocyanins and modulated ABA metabolism, leading to higher ABA-GE and 7¿OH-ABA and lower phaseic acid (PA) in berries compared to ¿M plants. Under the most stressful scenario (LD and 28/18¿°C), at harvest +M plants exhibited higher berry anthocyanins and 7¿OH-ABA and lower PA and dihydrophaseic acid (DPA) levels than ¿M plants. These findings highlight the involvement of ABA metabolism into the ability of AMF to improve some traits involved in the quality of grapes under global warming scenarios.

Background: The release of phytosiderephores (PS) to the rhizosphere is the main root response to iron (Fe) deficiency in graminaceous plants. We have investigated the role of the Fe status in the shoot as well as of the signaling pathways controlled by three relevant phytoregulators-indolacetic acid (IAA), ethylene and nitric oxide (NO) - in the regulation of this root response in Fe-starved wheat plants. To this end, the PS accumulation in the nutrient solution and the root expression of the genes encoding the nicotianamine aminotransferase (TaNAAT) and ferritin (TaFER) have been evaluated in plants subjected to different treatments. Results: The application of Fe to leaves of Fe-deficient plants prevented the increase in both PS root release and TaNAAT gene expression thus showing the relevant role of the shoot to root communication in the regulation of PS root release and some steps of PS biosynthesis. Experiments with specific hormone inhibitors showed that while ethylene and NO did not positively regulate Fe deficiency induced PS root release, auxin plays an essential role in the regulation of this process. Moreover, the application of IM to Fe-sufficient plants promoted both PS root release and TaNAAT gene expression thus indicating that auxin might be involved in the shoot to root signaling network regulating Fe-deficiency root responses in wheat Conclusions: These results therefore indicate that PS root release in Fe-deficient wheat plants is directly modulated by the shoot Fe status through signaling pathways involving, among other possible effectors, auxin.

The effect of ethylene and its precursor ACC on root hydraulic properties, including aquaporin expression and abundance, is modulated by relative air humidity and plant sensitivity to ethylene. Relative air humidity (RH) is a main factor contributing to water balance in plants. Ethylene (ET) is known to be involved in the regulation of root water uptake and stomatal opening although its role on plant water balance under different RH is not very well understood. We studied, at the physiological, hormonal and molecular levels (aquaporins expression, abundance and phosphorylation state), the plant responses to exogenous 1-aminocyclopropane-1-carboxylic acid (ACC; precursor of ET) and 2-aminoisobutyric acid (AIB; inhibitor of ET biosynthesis), after 24 h of application to the roots of tomato wild type (WT) plants and its ET-insensitive never ripe (nr) mutant, at two RH levels: regular (50%) and close to saturation RH. Highest RH induced an increase of root hydraulic conductivity (Lp(o)) of non-treated WT plants, and the opposite effect in nr mutants. The treatment with ACC reduced Lp(o) in WT plants at low RH and in nr plants at high RH. The application of AIB increased Lp(o) only in nr plants at high RH. In untreated plants, the RH treatment changed the abundance and phosphorylation of aquaporins that affected differently both genotypes according to their ET sensitivity. We show that RH is critical in regulating root hydraulic properties, and that Lp(o) is affected by the plant sensitivity to ET, and possibly to ACC, by regulating aquaporins expression and their phosphorylation status. These results incorporate the relationship between RH and ET in the response of Lp(o) to environmental changes.

Background and Aims Plant growth-promoting bacteria (PGPB) are soil micro-organisms able to interact with plants and stimulate their growth, positively affecting plant physiology and development. Although ethylene plays a key role in plant growth, little is known about the involvement of ethylene sensitivity in bacterial inoculation effects on plant physiology. Thus, the present study was pursued to establish whether ethylene perception is critical for plant-bacteria interaction and growth induction by two different PGPB strains, and to assess the physiological effects of these strains in juvenile and mature tomato (Solanum lycopersicum) plants. Methods An experiment was performed with the ethylene-insensitive tomato never ripe and its isogenic wildtype line in which these two strains were inoculated with either Bacillus megaterium or Enterobacter sp. C7. Plants were grown until juvenile and mature stages, when biomass, stomatal conductance, photosynthesis as well as nutritional, hormonal and metabolic statuses were analysed. Key Results Bacillus megaterium promoted growth only in mature wild type plants. However, Enterobacter C7 PGPB activity affected both wild-type and never ripe plants. Furthermore, PGPB inoculation affected physiological parameters and root metabolite levels in juvenile plants; meanwhile plant nutrition was highly dependent on ethylene sensitivity and was altered at the mature stage. Bacillus megaterium inoculation improved carbon assimilation in wild-type plants. However, insensitivity to ethylene compromised B. megaterium PGPB activity, affecting photosynthetic efficiency, plant nutrition and the root sugar content. Nevertheless, Enterobacter C7 inoculation modified the root amino acid content in addition to stomatal conductance and plant nutrition. Conclusions Insensitivity to ethylene severely impaired B. megaterium interaction with tomato plants, resulting in physiological modifications and loss of PGPB activity. In contrast, Enterobacter C7 inoculation stimulated growth independently of ethylene perception and improved nitrogen assimilation in ethylene-insensitive plants. Thus, ethylene sensitivity is a determinant for B. megaterium, but is not involved in Enterobacter C7 PGPB activity.

The use of plant growth promoting rhizobacteria (PGPR) is a proven management strategy to improve plant growth. The aim was to reveal the genomic and proteomic basis of the plant tolerance to saline soil conditions. Combination of whole transcriptome analysis and proteomic profiling helped further the understanding of the complexity of salt tolerance responses. Arabidopsis plants were grown inoculated or not with Bacillus megaterium and irrigated or not with salt. Physiological, genomic and proteomic approaches were combined to reveal plant salt tolerance mechanisms. Microarray analyses revealed the up-regulation of the jasmonic acid metabolism (CYP94B3, lipooxigenase 4 and allene-oxide cyclase) under saline conditions. Knock-out mutants of the gene of interest CYP94B3, responsible of JA-Ile catabolism, were used to confirm the obtained results. Salinity resulted in leaf Na accumulation with decreased chlorophyll content, but PGPR inoculation helped to overcome the stress. Proteomic analysis showed enhanced monodehydroascorbate reductase (MDHAR) content together with ATP synthase. CYP94B3 knock-out plants confirmed the key role of JA-Ile turnover to overcome moderate saline stress. Subsequent experimentation showed that CYP94B3 was important for salt tolerance under moderate and severe salt stress. Inoculation with B. megaterium was a valuable tool to reveal the importance of JA-Ile turnover and to recover Arabidopsis plants from saline stress.

Hormonal regulation and symbiotic relationships provide benefits for plants to overcome stress conditions. The aim of this study was to elucidate the effects of exogenous methyl jasmonate (MeJA) application on root hydraulic conductivity (L) of Phaseolus vulgaris plants which established arbuscular mycorrhizal (AM) symbiosis under two water regimes (well-watered and drought conditions). The variation in endogenous contents of several hormones (MeJA, JA, abscisic acid (ABA), indol-3-acetic acid (IAA), salicylic acid (SA)) and the changes in aquaporin gene expression, protein abundance and phosphorylation state were analyzed. AM symbiosis decreased L under well-watered conditions, which was partially reverted by the MeJA treatment, apparently by a drop in root IAA contents. Also, AM symbiosis and MeJA prevented inhibition of L under drought conditions, most probably by a reduction in root SA contents. Additionally, the gene expression of two fungal aquaporins was upregulated under drought conditions, independently of the MeJA treatment. Plant aquaporin gene expression could not explain the behaviour of L. Conversely, evidence was found for the control of L by phosphorylation of aquaporins. Hence, MeJA addition modified the response of L to both AM symbiosis and drought, presumably by regulating the root contents of IAA and SA and the phosphorylation state of aquaporins.

Arbuscular mycorrhizal (AM) symbiosis alleviates drought stress in plants. However, the intimate mechanisms involved, as well as its effect on the production of signalling molecules associated with the host plant-AM fungus interaction remains largely unknown. In the present work, the effects of drought on lettuce and tomato plant performance and hormone levels were investigated in non-AM and AM plants. Three different water regimes were applied, and their effects were analysed over time. AM plants showed an improved growth rate and efficiency of photosystem II than non-AM plants under drought from very early stages of plant colonization. The levels of the phytohormone abscisic acid, as well as the expression of the corresponding marker genes, were influenced by drought stress in non-AM and AM plants. The levels of strigolactones and the expression of corresponding marker genes were affected by both AM symbiosis and drought. The results suggest that AM symbiosis alleviates drought stress by altering the hormonal profiles and affecting plant physiology in the host plant. In addition, a correlation between AM root colonization, strigolactone levels and drought severity is shown, suggesting that under these unfavourable conditions, plants might increase strigolactone production in order to promote symbiosis establishment to cope with the stress.

We aimed to identify the early stress response and plant performance of Medicago truncatula growing in axenic medium with ammonium or urea as the sole source of nitrogen, with respect to nitrate-based nutrition. Biomass measurements, auxin content analyses, root system architecture (RSA) response analyses, and physiological parameters were determined. Both ammonium and ureic nutrition severely affected the RSA, resulting in changes in the main elongation rate, lateral root development, and insert position from the root base. The auxin content decreased in both urea- and ammonium-treated roots; however, only the ammonium-treated plants were affected at the shoot level. The analysis of chlorophyll a fluorescence transients showed that ammonium affected photosystem II, but urea did not impair photosynthetic activity. Superoxide dismutase isoenzymes in the plastids were moderately affected by urea and ammonium in the roots. Overall, our results showed that low N doses from different sources had no remarkable effects on M. truncatula, with the exception of the differential phenotypic root response. High doses of both ammonium and urea caused great changes in plant length, auxin contents and physiological measurements. Interesting correlations were found between the shoot auxin pool and both plant length and the "performance index" parameter, which is obtained from measurements of the kinetics of chlorophyll a fluorescence.

The presence of arsenic in soil and water is a constant threat to plant growth in many regions of the world. Phytohormones act in the integration of growth control and stress response, but their role in plant responses to arsenic remains to be elucidated. Here, we show that arsenate [As(V)], the most prevalent arsenic chemical species in nature, causes severe depletion of endogenous cytokinins (CKs) in the model plant Arabidopsis (Arabidopsis thaliana). We found that CK signaling mutants and transgenic plants with reduced endogenous CK levels showed an As(V)-tolerant phenotype. Our data indicate that in CK-depleted plants exposed to As(V), transcript levels of As(V)/phosphate-transporters were similar or even higher than in wild-type plants. In contrast, CK depletion provoked the coordinated activation of As(V) tolerance mechanisms, leading to the accumulation of thiol compounds such as phytochelatins and glutathione, which are essential for arsenic sequestration. Transgenic CK-deficient Arabidopsis and tobacco lines show a marked increase in arsenic accumulation. Our findings indicate that CK is an important regulatory factor in plant adaptation to arsenic stress.

The physiological and metabolic mechanisms behind the humic acid-mediated plant growth enhancement are discussed in detail. Experiments using cucumber (Cucumis sativus) plants show that the shoot growth enhancement caused by a structurally well-characterized humic acid with sedimentary origin is functionally associated with significant increases in abscisic acid (ABA) root concentration and root hydraulic conductivity. Complementary experiments involving a blocking agent of cell wall pores and water root transport (polyethylenglycol) show that increases in root hydraulic conductivity are essential in the shoot growth-promoting action of the model humic acid. Further experiments involving an inhibitor of ABA biosynthesis in root and shoot (fluridone) show that the humic acid-mediated enhancement of both root hydraulic conductivity and shoot growth depended on ABA signaling pathways. These experiments also show that a significant increase in the gene expression of the main root plasma membrane aquaporins is associated with the increase of root hydraulic conductivity caused by the model humic acid. Finally, experimental data suggest that all of these actions of model humic acid on root functionality, which are linked to its beneficial action on plant shoot growth, are likely related to the conformational structure of humic acid in solution and its interaction with the cell wall at the root surface.

Identification of early sulphur (S) deficiency indicators is important for species such as Brassica napus, an S-demanding crop in which yield and the nutritional quality of seeds are negatively affected by S deficiency. Because S is mostly stored as SO42- in leaf cell vacuoles and can be mobilized during S deficiency, this study investigated the impact of S deprivation on leaf osmotic potential in order to identify compensation processes. Plants were exposed for 28 days to S or to chlorine deprivation in order to differentiate osmotic and metabolic responses. While chlorine deprivation had no significant effects on growth, osmotic potential and nitrogen metabolism, Brassica napus revealed two response periods to S deprivation. The first one occurred during the first 13 days during which plant growth was maintained as a result of vacuolar SO42- mobilization. In the meantime, leaf osmotic potential of S-deprived plants remained similar to control plants despite a reduction in the SO42- osmotic contribution, which was fully compensated by an increase in NO3-, PO43- and Cl- accumulation. The second response occurred after 13 days of S deprivation with a significant reduction in growth, leaf osmotic potential, NO3- uptake and NO3- reductase activity, whereas amino acids and NO3- were accumulated. This kinetic analysis of S deprivation suggested that a ([Cl-]+[NO3-]+[PO43-]):[SO42-] ratio could provide a relevant indicator of S deficiency,

Although climate scenarios have predicted an increase in [CO2] and temperature conditions, to date few experiments have focused on the interaction of [CO2] and temperature effects in wheat development. Recent evidence suggests that photosynthetic acclimation is linked to the photorespiration and N assimilation inhibition of plants exposed to elevated CO2. The main goal of this study was to analyze the effect of interacting [CO2] and temperature on leaf photorespiration, C/N metabolism and N transport in wheat plants exposed to elevated [CO2] and temperature conditions. For this purpose, wheat plants were exposed to elevated [CO2] (400 vs 700 mu molmol(-1)) and temperature (ambient vs ambient+4 degrees C) in CO2 gradient greenhouses during the entire life cycle. Although at the agronomic level, elevated temperature had no effect on plant biomass, physiological analyses revealed that combined elevated [CO2] and temperature negatively affected photosynthetic performance. The limited energy levels resulting from the reduced respiratory and photorespiration rates of such plants were apparently inadequate to sustain nitrate reductase activity. Inhibited N assimilation was associated with a strong reduction in amino acid content, conditioned leaf soluble protein content and constrained leaf N status. Therefore, the plant response to elevated [CO2] and elevated temperature resulted in photosynthetic acclimation. The reduction in transpiration rates induced limitations in nutrient transport in leaves of plants exposed to elevated [CO2] and temperature, led to mineral depletion and therefore contributed to the inhibition of photosynthetic activity.

Although climate scenarios have predicted an increase in [CO2] and temperature conditions, to date few experiments have focused on the interaction of [CO2] and temperature effects in wheat development. Recent evidence suggests that photosynthetic acclimation is linked to the photorespiration and N assimilation inhibition of plants exposed to elevated CO2. The main goal of this study was to analyze the effect of interacting [CO2] and temperature on leaf photorespiration, C/N metabolism and N transport in wheat plants exposed to elevated [CO2] and temperature conditions. For this purpose, wheat plants were exposed to elevated [CO2] (400 vs 700 mu molmol(-1)) and temperature (ambient vs ambient+4 degrees C) in CO2 gradient greenhouses during the entire life cycle. Although at the agronomic level, elevated temperature had no effect on plant biomass, physiological analyses revealed that combined elevated [CO2] and temperature negatively affected photosynthetic performance. The limited energy levels resulting from the reduced respiratory and photorespiration rates of such plants were apparently inadequate to sustain nitrate reductase activity. Inhibited N assimilation was associated with a strong reduction in amino acid content, conditioned leaf soluble protein content and constrained leaf N status. Therefore, the plant response to elevated [CO2] and elevated temperature resulted in photosynthetic acclimation. The reduction in transpiration rates induced limitations in nutrient ...

The role of jasmonic acid in the induction of stomatal closure is well known. However, its role in regulating root hydraulic conductivity (L) has not yet been explored. The objectives of the present research were to evaluate how JA regulates L and how calcium and abscisic acid (ABA) could be involved in such regulation. We found that exogenous methyl jasmonate (MeJA) increased L of Phaseolus vulgaris, Solanum lycopersicum and Arabidopsis thaliana roots. Tomato plants defective in JA biosynthesis had lower values of L than wild-type plants, and that L was restored by addition of MeJA. The increase of L by MeJA was accompanied by an increase of the phosphorylation state of the aquaporin PIP2. We observed that MeJA addition increased the concentration of cytosolic calcium and that calcium channel blockers inhibited the rise of L caused by MeJA. Treatment with fluoridone, an inhibitor of ABA biosynthesis, partially inhibited the increase of L caused by MeJA, and tomato plants defective in ABA biosynthesis increased their L after application of MeJA. It is concluded that JA enhances L and that this enhancement is linked to calcium and ABA dependent and independent signalling pathways.

BACKGROUND: Plant growth-promoting rhizobacteria (PGPR) are naturally occurring soil bacteria which benefit plants by improving plant productivity and immunity. The mechanisms involved in these processes include the regulation of plant hormone levels such as ethylene and abscisic acid (ABA). The aim of the present study was to determine whether the activity of Bacillus megaterium PGPR is affected by the endogenous ABA content of the host plant. The ABA-deficient tomato mutants flacca and sitiens and their near-isogenic wild-type parental lines were used. Growth, stomatal conductance, shoot hormone concentration, competition assay for colonization of tomato root tips, and root expression of plant genes expected to be modulated by ABA and PGPR were examined. RESULTS: Contrary to the wild-type plants in which PGPR stimulated growth rates, PGPR caused growth inhibition in ABA-deficient mutant plants. PGPR also triggered an over accumulation of ethylene in ABA-deficient plants which correlated with a higher expression of the pathogenesis-related gene Sl-PR1b. CONCLUSIONS: Positive correlation between over-accumulation of ethylene and a higher expression of Sl-PR1b in ABA-deficient mutant plants could indicate that maintenance of normal plant endogenous ABA content may be essential for the growth promoting action of B. megaterium by keeping low levels of ethylene production.

Previous studies have reported that a purified sedimentary humic acid (PHA) was able to increase the concentration of nitric oxide (NO), indole-acetic acid (IAA) and ethylene in cucumber roots. Here, we investigated if these effects are functionally related to the ability of PHA to improve shoot growth. The effect of specific inhibitors of NO, IAA and ethylene functionality and signaling on PHA-induced shoot growth was studied. Likewise, the effect of these inhibitors on the synthesis and activity of the phytoregulators concerned by PHA action in cucumber roots was also explored. The results show that shoot growth promoted by PHA is due to an increase of IAA concentration in the root through both a NO-dependent and a NO-independent pathway. In addition, the increased ethylene production in the root is regulated by an IAA-dependent pathway. Finally, results also showed that the increase of ABA concentration in the root is regulated through both IAA- and ethylene-dependent pathways. In summary, the shoot growth promoting action of PHA involves a complex hormonal network. On one hand, the PHA action is functionally linked to increments in NO and IAA concentration in roots. And on the other hand, PHA action also increases ethylene and ABA root concentration mediated by NO-IAA dependent pathways.

It is known that the presence of arbuscular mycorrhizal fungi within the plant roots enhances the tolerance of the host plant to different environmental stresses, although the positive effect of the fungi in plants under waterlogged conditions has not been well studied. Tolerance of plants to flooding can be achieved through different molecular, physiological and anatomical adaptations, which will affect their water uptake capacity and therefore their root hydraulic properties. Here, we investigated the root hydraulic properties under non-flooded and flooded conditions in non-mycorrhizal tomato plants and plants inoculated with the arbuscular mycorrhizal fungus Rhizophagus irregularis. Only flooded mycorrhizal plants increased their root hydraulic conductivity, and this effect was correlated with a higher expression of the plant aquaporin SlPIP1;7 and the fungal aquaporin GintAQP1. There was also a higher abundance of the PIP2 protein phoshorylated at Ser280 in mycorrhizal flooded plants. The role of plant hormones (ethylene, ABA and IAA) in root hydraulic properties was also taken into consideration, and it was concluded that, in mycorrhizal flooded plants, ethylene has a secondary role regulating root hydraulic conductivity whereas IAA may be the key hormone that allows the enhancement of root hydraulic conductivity in mycorrhizal plants under low oxygen conditions.

Arbuscular mycorrhizal (AM) symbiosis can alleviate salt stress in plants. However the intimate mechanisms involved, as well as the effect of salinity on the production of signalling molecules associated to the host plant-AM fungus interaction remains largely unknown. In the present work, we have investigated the effects of salinity on lettuce plant performance and production of strigolactones, and assessed its influence on mycorrhizal root colonization. Three different salt concentrations were applied to mycorrhizal and non-mycorrhizal plants, and their effects, overtime, analyzed. Plant biomass, stomatal conductance, efficiency of photosystem II, as well as ABA content and strigolactone production were assessed. The expression of ABA biosynthesis genes was also analyzed. AM plants showed improved growth rates and a better performance of physiological parameters such as stomatal conductance and efficiency of photosystem II than non-mycorrhizal plants under salt stress since very early stages - 3 weeks - of plant colonization. Moreover, ABA levels were lower in those plants, suggesting that they were less stressed than non-colonized plants. On the other hand, we show that both AM symbiosis and salinity influence strigolactone production, although in a different way in AM and non-AM plants. The results suggest that AM symbiosis alleviates salt stress by altering the hormonal. profiles and affecting plant physiology in the host plant.

An understanding of the mechanisms underlying ammonium (NH4+) toxicity in plants requires prior knowledge of the metabolic uses for nitrogen (N) and carbon (C). We have recently shown that pea plants grown at high NH4+ concentrations suffer an energy deficiency associated with a disruption of ionic homeostasis. Furthermore, these plants are unable to adequately regulate internal NH4+ levels and the cell-charge balance associated with cation uptake. Herein we show a role for an extra-C application in the regulation of C¿N metabolism in NH4+-fed plants. Thus, pea plants (Pisum sativum) were grown at a range of NH4+ concentrations as sole N source, and two light intensities were applied to vary the C supply to the plants. Control plants grown at high NH4+ concentration triggered a toxicity response with the characteristic pattern of C-starvation conditions. This toxicity response resulted in the redistribution of N from amino acids, mostly asparagine, and lower C/N ratios. The C/N imbalance at high NH4+ concentration under control conditions induced a strong activation of root C metabolism and the upregulation of anaplerotic enzymes to provide C intermediates for the tricarboxylic acid cycle. A high light intensity partially reverted these C-starvation symptoms by providing higher C availability to the plants. The extra-C contributed to a lower C4/C5 amino acid ratio while maintaining the relative contents of some minor amino acids involved in key pathways regulating the C/N sta

Iron homeostasis is an important process for flower development and plant fertility. The role of plastids in these processes has been shown to be essential. To document the relationships between plastid iron homeostasis and flower biology further, a global study (transcriptome, proteome, metabolome, and hormone analysis) was performed of Arabidopsis flowers from wild-type and triple atfer1-3-4 ferritin mutant plants grown under iron-sufficient or excess conditions. Some major modifications in specific functional categories were consistently observed at these three omic levels, although no significant overlaps of specific transcripts and proteins were detected. These modifications concerned redox reactions and oxidative stress, as well as amino acid and protein catabolism, this latter point being exemplified by an almost 10-fold increase in urea concentration of atfer1-3-4 flowers from plants grown under iron excess conditions. The mutant background caused alterations in Fe-haem redox proteins located in membranes and in hormone-responsive proteins. Specific effects of excess Fe in the mutant included further changes in these categories, supporting the idea that the mutant is facing a more intense Fe/redox stress than the wild type. The mutation and/or excess Fe had a strong impact at the membrane level, as denoted by the changes in the transporter and lipid metabolism categories.

The main objective of this study is to investigate the more relevant binding sites (functional groups) involved in the complexation of Fe(III) and Cu(II) by humic-based structures. To this aim we have generated a set of seven humic-based samples (MHA) by the application of a hemi-synthetic process consisting in the oxidative polymerization of different combinations of three sources of natural organic molecules: two humic acids obtained from peat and leonardite, and natural tannin extracted from the quebracho tree (Schinopsis sp.) bark. These MHA were extensively characterized by using complementary analytical techniques (13C NMR, size-exclusion chromatography, electron paramagnetic resonance, and total phenol-reductant content), and their ability to complex Fe(III) and Cu(II) was also calculated by fluorescence quenching method. The data were studied by using general correlation matrix and principal component analysis. The results obtained, taken together, indicated that in a set of humic-based structures presenting carboxylic, phenol and O-alkyl groups, and diverse aliphatic/aromatic character, the complexation of Fe(III) mainly involved specific structural arrangements including carboxylic groups distributed in aliphatic domains. However, Cu(II) complexation involved singular structural arrangements including phenols (and probably substituted phenols) and O-alkyl groups in side chains of aromatic domains.

The increase in root growth is one of the major effects of humic substances, but the mechanisms involved in humic acid-mediated changes in root growth, morphology and architecture are poorly known. Probably, humic substances may act on plant development through an action on the hormonal balance within the plant, either directly or indirectly by affecting the root uptake of some nutrients. In this study we investigate in cucumber plants the effects of a purified sedimentary humic acid (PHA), without detectable concentrations of the main phytoregulators in its structure, on root architecture, and its relationships with the functional action of three phytoregulators, indole-acetic acid, ethylene and nitric oxide, which are also affected by the root application of this humic acid. The results obtained using inhibitors of auxin transport or action, inhibitors of ethylene biosynthesis or action, and a scavenger of nitric oxide indicate that the increase in the root concentration of these phytoregulators caused by the root application of PHA does not play an essential role in the expression of the main changes on root architecture caused by PHA in developed cucumber plants. Other factors, which could act in coordination or independently of those phytoregulators affected by PHA root application, must be involved in the whole action on this humic acid on root architecture in cucumber.

The aim of this study was to investigate the effects of IAA and ABA in the shoot-to-root regulation of the expression of the main Fe-stress physiological root responses in cucumber plants subjected to shoot Fe functional deficiency. Changes in the expression of the genes CsFRO1, CsIRT1, CsHA1 and CsHA2 (coding for Fe(III)-chelate reductase (FCR), the Fe(II) transporter and H+-ATPase, respectively) and in the enzyme activity of FCR and the acidification capacity were measured. We studied first the ability of exogenous applications of IAA and ABA to induce these Fe-stress root responses in plants grown in Fe-sufficient conditions. The results showed that IAA was able to activate these responses at the transcriptional and functional levels, whereas the results with ABA were less conclusive. Thereafter, we explored the role of IAA in plants with or without shoot Fe functional deficiency in the presence of two types of IAA inhibitors, affecting either IAA polar transport (TIBA) or IAA functionality (PCIB). The results showed that IAA is involved in the regulation at the transcriptional and functional levels of both Fe root acquisition (FCR, Fe(II) transport) and rhizosphere acidification (H+-ATPase), although through different, and probably complementary, mechanisms. These results suggest that IAA is involved in the shoot-to-root regulation of the expression of Fe-stress physiological root responses.

Numerous studies have reported the ability of humic substances to increase shoot growth in different plant species cultivated under diverse growth conditions. However, the mechanism responsible for this effect of humic substances is poorly understood. It is possible that the shoot promoting effect of humic substances involves a primary effect on root H(+)-ATPase activity and nitrate root-shoot distribution that, in turn, causes changes in the root-shoot distribution of certain cytokinins, polyamines and abscisic acid, thus affecting shoot growth. We investigated this hypothesis in the present study. The results showed that the root application of a purified humic acid causes a significant increase in shoot growth that is associated with an enhancement in root H(+)-ATPase activity, an increase in nitrate shoot concentration, and a decrease in roots. These effects were associated with significant increases in the shoot concentration of several cytokinins and polyamines (principally putrescine), concomitant with decreases in roots. Likewise, these changes in the root-shoot distribution of diverse active cytokinins correlated well to significant changes in the root-shoot distribution of several mineral nutrients. These results, taken together, indicate that the beneficial effects of humic substances on shoot development in cucumber could be directly associated with nitrate-related effects on the shoot concentration of several active cytokinins and polyamines (principally putrescine).

BACKGROUND: In certain plant species, ammonium or urea nutrition can cause negative effects on plant development which can result in toxic symptoms. Some authors suggest that the presence of nitrate can alleviate these symptoms by increasing ammonium and urea assimilation, avoiding its accumulation. In order to study this hypothesis, wheat (Triticum aestivum L.) seedlings were grown with various nitrogen supplies containing the main nitrogen forms (ammonium, nitrate and urea). Amino acids content and the activity of the three main enzymes involved in nitrogen assimilation (nitrate reductase, glutamine synthetase and urease) were studied. RESULTS: The application of nitrate along with urea and/or ammonium was not associated with a time¿sustained increase in the activity of glutamine synthetase and urease. Amino acid analysis revealed that nitrate induced changes in amino acid metabolism enhancing its concentration. Likewise the content of protein was also higher in nitrate¿treated plants. CONCLUSION: These results suggest that the effect of nitrate is compatible with a rapid and transient increase in the activity of glutamine synthetase and urease during the first hour after the onset of treatments. Nevertheless, a possible effect of nitrate reducing ammonium accumulation through the activation of alternative metabolic pathways different from that involving glutamine synthetase cannot be ruled out.