Interaction of ammonium nutrition with essential mineral cations.

Plant growth and development depend on sufficient nutrient availability in soils. Agricultural soils are generally nitrogen (N) deficient, and thus soils need to be supplemented with fertilizers. Ammonium (NH4+) is a major inorganic N source. However, at high concentrations, NH4+ becomes a stressor that inhibits plant growth. The cause of NH4+ stress or toxicity is multifactorial, but the interaction of NH4+ with other nutrients is among the main determinants of plants' sensitivity towards high NH4+ supply. In addition, NH4+ uptake and assimilation provoke the acidification of the cell external medium (apoplast/rhizosphere), which has a clear impact on nutrient availability. This review summarizes current knowledge, at both the physiological and the molecular level, of the interaction of NH4+ nutrition with essential mineral elements that are absorbed as cations, both macronutrients (K+, Ca2+, Mg2+) and micronutrients (Fe2+/3+, Mn2+, Cu+/2+, Zn2+, Ni2+). We hypothesize that considering these nutritional interactions, and soil pH, when formulating fertilizers may be key in order to boost the use of NH4+-based fertilizers, which have less environmental impact compared with nitrate-based ones. In addition, we are convinced that better understanding of these interactions will help to identify novel targets with the potential to improve crop productivity.

Exploiting Tomato Genotypes to Understand Heat Stress Tolerance.

Increased temperatures caused by climate change constitute a significant threat to agriculture and food security. The selection of improved crop varieties with greater tolerance to heat stress is crucial for the future of agriculture. To overcome this challenge, four traditional tomato varieties from the Mediterranean basin and two commercial genotypes were selected to characterize their responses at high temperatures. The screening of phenotypes under heat shock conditions allowed to classify the tomato genotypes as: heat-sensitive: TH-30, ADX2; intermediate: ISR-10 and Ailsa Craig; heat-tolerant: MM and MO-10. These results reveal the intra-genetical variation of heat stress responses, which can be exploited as promising sources of tolerance to climate change conditions. Two different thermotolerance strategies were observed. The MO-10 plants tolerance was based on the control of the leaf cooling mechanism and the rapid RBOHB activation and ABA signaling pathways. The variety MM displayed a different strategy based on the activation of HSP70 and 90, as well as accumulation of phenolic compounds correlated with early induction of PAL expression. The importance of secondary metabolism in the recovery phase has been also revealed. Understanding the molecular events allowing plants to overcome heat stress constitutes a promising approach for selecting climate resilient tomato varieties.

Physiological and Antioxidant Response to Different Water Deficit Regimes of Flag Leaves and Ears of Wheat Grown under Combined Elevated CO2 and High Temperature.

Triticum aestivum L. cv. Gazul is a spring wheat widely cultivated in Castilla y León (Spain). Potted plants were grown in a scenario emulating the climate change environmental conditions expected by the end of this century, i.e., with elevated CO2 and high temperature under two water deficit regimes: long (LWD) and terminal (TWD). Changes in biomass and morphology, the content of proline (Pro), ascorbate (AsA) and glutathione (GSH), and enzymatic antioxidant activities were analyzed in flag leaves and ears. Additionally, leaf gas exchange was measured. LWD caused a decrease in biomass and AsA content but an increase in Pro content and catalase and GSH reductase activities in flag leaves, whereas TWD produced no significant changes. Photosynthesis was enhanced under both water deficit regimes. Increase in superoxide dismutase activity and Pro content was only observed in ears under TWD. The lack of a more acute effect of LWD and TWD on both organs was attributed to the ROS relieving effect of elevated CO2. Gazul acted as a drought tolerant variety with anisohydric behavior. A multifactorial analysis showed better adaptation of ears to water deficit than flag leaves, underlining the importance of this finding for breeding programs to improve grain yield under future climate change.

Putrescine: A Key Metabolite Involved in Plant Development, Tolerance and Resistance Responses to Stress.

Putrescine (Put) is the starting point of the polyamines (PAs) pathway and the most common PA in higher plants. It is synthesized by two main pathways (from ornithine and arginine), but recently a third pathway from citrulline was reported in sesame plants. There is strong evidence that Put may play a crucial role not only in plant growth and development but also in the tolerance responses to the major stresses affecting crop production. The main strategies to investigate the involvement of PA in plant systems are based on the application of competitive inhibitors, exogenous PAs treatments, and the most efficient approaches based on mutant and transgenic plants. Thus, in this article, the recent advances in understanding the role of this metabolite in plant growth promotion and protection against abiotic and biotic stresses will be discussed to provide an overview for future research.

Putrescine biosynthetic pathways modulate root growth differently in tomato seedlings grown under different N sources.

The biosynthesis of putrescine is mainly driven by arginine decarboxylase (ADC) and ornithine decarboxylase (ODC). Hence, in this study, we generated independent ADC and ODC transgenic silenced tomato lines (SilADC and SilODC, respectively) to test the effect of defective ADC and ODC gene expression on root development under nitrate (NN) or ammonium (NA) conditions. The results showed that SilODC seedlings displayed an increase in ADC expression that led to polyamine accumulation, suggesting a compensatory effect of ADC. However, this effect was not observed in SilADC seedlings. These pathways are involved in different growth processes. The SilADC seedlings showed an increase in fresh weight, shoot length, lateral root number and shoot:root ratio under the NN source and an enhancement in fresh weight, and shoot and root length under NA conditions. However, SilODC seedlings displayed greater weight and shoot length under the NN source, whereas a decrease in lateral root density was found under NA conditions. Moreover, two overexpressed ODC lines were generated to check the relevance of the compensatory effect of the ADC pathway when ODC was silenced. These overexpressed lines showed not only an enhancement of almost all the studied growth parameters under both N sources but also an amelioration of ammonium syndrome under NA conditions. Together, these results reflect the importance of both pathways in plant growth, particularly ODC silencing, which requires compensation by ADC induction.

Theoretical and Experimental Considerations for a Rapid and High Throughput Measurement of Catalase In Vitro.

A rapid and high throughput protocol to measure the catalase activity in vitro has been designed. Catalase is an enzyme with unusual kinetic properties because it does not follow the standard Michaelis-Menten model and is inactivated by H2O2. This makes the analysis of the two rate equations of the second-ordered reactions of the kinetic model rather complex. A two-degree polynomial fitting of the experimental data is proposed after transforming the exponential form of the integrated rate equation of the [H2O2] into a polynomial using the Taylor series. The fitting is validated by establishing an experimental linear relationship between the initial rate of the H2O2 decomposition and the protein concentration, regardless of the suicide inactivation that catalase might undergo beyond t > 0. In addition, experimental considerations are taken into account to avoid statistical bias in the analysis of the catalase activity. ANOVA analyses show that the proposed protocol can be utilized to measure the initial rate of the H2O2 decomposition by catalase in 32 samples in triplicates if kept below 8 mM min-1 in the microplate wells. These kinetic and statistical analyses can pave the way for other antioxidant enzyme activity assays in microplate readers at small scale and low cost.

1-Methyltryptophan Treatment Increases Defense-Related Proteins in the Apoplast of Tomato Plants.

The activation of induced resistance in plants may enhance the production of defensive proteins to avoid the invasion of pathogens. In this way, the composition of the apoplastic fluid could represent an important layer of defense that plants can modify to avoid the attack. In this study, we performed a proteomic study of the apoplastic fluid from plants treated with the resistance inducer 1-methyltryptophan (1-MT) as well as infected with Pseudomonas syringae pv. tomato (Pst). Our results showed that both the inoculation with Pst and the application of the inducer provoke changes in the proteomic composition in the apoplast enhancing the accumulation of proteins involved in plant defense. Finally, one of the identified proteins that are overaccumulated upon the treatment have been expressed in Escherichia coli and purified in order to test their antimicrobial effect. The result showed that the tested protein is able to reduce the growth of Pst in vitro. Taken together, in this work, we described the proteomic changes in the apoplast induced by the treatment and by the inoculation, as well as demonstrated that the proteins identified have a role in the plant protection.

Exogenous Carbon Compounds Modulate Tomato Root Development.

NO3- is not only a nutrient, but also a signaling compound that plays an important role in several plant processes, like root development. The present study aimed to investigate the effect of three different exogenous C compounds (sucrose, glucose, 2-oxoglutarate) added to NO3- nutrition on C/N, auxin and antioxidant metabolisms in 10-day-old tomato seedlings. Sucrose and glucose supplementation enhanced primary root (PR) length, lateral root number and root density, while 2-oxoglutarate negatively affected them. This phenomenon was accompanied by a slight increase in NRT2.1 and GS1 gene expression, together with an increase in LAX2 and LAX3 and a decrease in LAX4 in the roots growing under sucrose and glucose sources. The addition of 2-oxoglutarate enhanced the expression of NiR, GDH, PEPC1, LAX1, LAX3 and the antioxidant gene SOD Cl. Taken together, these findings contribute to a better understanding of how these C sources can modulate N uptake and C/N, auxin and antioxidant gene expression, which could be useful for improving nitrogen use efficiency.

The Apoplast: A Key Player in Plant Survival.

The apoplast comprises the intercellular space, the cell walls, and the xylem. Important functions for the plant, such as nutrient and water transport, cellulose synthesis, and the synthesis of molecules involved in plant defense against both biotic and abiotic stresses, take place in it. The most important molecules are ROS, antioxidants, proteins, and hormones. Even though only a small quantity of ROS is localized within the apoplast, apoplastic ROS have an important role in plant development and plant responses to various stress conditions. In the apoplast, like in the intracellular cell compartments, a specific set of antioxidants can be found that can detoxify the different types of ROS produced in it. These scavenging ROS components confer stress tolerance and avoid cellular damage. Moreover, the production and accumulation of proteins and peptides in the apoplast take place in response to various stresses. Hormones are also present in the apoplast where they perform important functions. In addition, the apoplast is also the space where microbe-associated molecular Patterns (MAMPs) are secreted by pathogens. In summary, the diversity of molecules found in the apoplast highlights its importance in the survival of plant cells.

Standardization of a Culture Medium for Megaselia scalaris (Diptera: Phoridae) for Entomotoxicological Studies.

One of the problems faced by entomotoxicological studies is the large variability of experimental set-ups and the absence of harmonized protocols to compare the data and results obtained by different research groups. Among the wide range of influencing factors on the development and growth of insects, food substrates are remarkably relevant. This article proposes a standardized growth medium to be employed in future entomotoxicological studies on the scuttle fly Megaselia scalaris (Loew, 1866), (Diptera: Phoridae). This species plays an important role in forensic cases related to the decomposition of human remains found indoors and/or in concealed environments, because of their small size and ability to enter these locations at an earlier time than many other insects. Because of these traits, it can be used for the estimation of the minimum postmortem interval (mPMI). We achieved the formulation of a medium that overcomes two traditional disadvantages of culture media used to raise necrophagous Diptera, the unknown media composition and different growth rates from those reared in tissue. The proposed medium is a known composition formulation, free of xenobiotics, in which M. scalaris shows growth rates and development times similar to those obtained when it is reared in tissue. This new diet might be used in future studies to test the effect of substances of forensic interest (prescription or illicit drugs, poisons, etc.) on the larval development time, helping to adjust the estimation of mPMI based on the presence of such substances. Additionally, the formulation revealed some interesting data about nutritional requirements of this species.

Condom Use Errors and Problems in a Sample of Young Colombian Adults.

Condom effectiveness can be reduced by a number of problems and errors that take place during use. The purpose of the present study was to translate and adapt the Condom Use Errors and Problems Survey (CUES) into Spanish and to use it for testing the prevalence of these issues in a sample of young Colombian adults. A total of 775 people (414 women and 361 men) between 18 and 26 years of age participated in the study. Participants completed the following questionnaires online: Condom Use Errors and Problems Survey, Sexual Opinion Survey, Sexual Assertiveness Scale, Multidimensional Condom Attitudes Scale, a sociodemographic and sexual history questionnaire, plus two questions concerning condom-associated erectile problems. Results showed significant differences in the prevalence of condom use errors/problems associated with gender, sexual orientation, and whether the participant had a stable sexual partner. The occurrence of problems/errors ranged from 9.4% to 69.5%. Except for certain specific practices, observations were similar to those of studies performed in North America. Results indicate that the present linguistic adaptation of the CUES for its use in Colombian Spanish-speaking populations is an effective instrument to assess problems and errors associated with the use of male condoms.

Tomato root development and N assimilation depend on C and ABA content under different N sources.

Root plasticity is controlled by hormonal homeostasis and nutrient availability. In this work, we have determined the influence of different N regimens on growth parameters and on the expression of genes involved in auxin transport and N-assimilation in tomato seedlings. NH4+ nutrition led to an inhibitory effect on root fresh weight (FW), lateral root (LR) number and root density, while an increase in the primary root (PR) length was observed. The expression of N assimilation genes GS2 and ASN1, is affected by NH4+ nutrition. Moreover, in order to relieve the toxic effect of NH4+ on root development, glucose or 2-oxoglutarate was supplied as a C source during NH4+ treatment. The addition of 2-oxoglutarate improved root parameters compared to the NH4+ regimen. N-assimilation gene analysis showed that NH4+-fed tomato plants try to alleviate the toxic effect by concurrently upregulating ASN1 and anaplerotic PEPC2 expression, whereas when 2-oxoglutarate is supplied, ASN1 induction was not observed. The addition of both C skeletons induced the expression of the ROS-scavenging genes GSH and SOD. In addition, since ABA plays a role in root development, the ABA-synthesis-defective mutant flacca was studied under NO3- and NH4+ regimens. It displayed a decrease in LR number under NO3- conditions, whereas, the NH4+-fed seedlings showed a decrease solely in PR length that was reverted when ABA was exogenously supplied. Moreover, flacca seedlings displayed a reprogramming of the N/C assimilation genes. Altogether, these results reflect the importance of N and C sources and ABA homeostasis in root development of tomato seedlings.

Expression of miR159 Is Altered in Tomato Plants Undergoing Drought Stress.

In a scenario of global climate change, water scarcity is a major threat for agriculture, severely limiting crop yields. Therefore, alternatives are urgently needed for improving plant adaptation to drought stress. Among them, gene expression reprogramming by microRNAs (miRNAs) might offer a biotechnologically sound strategy. Drought-responsive miRNAs have been reported in many plant species, and some of them are known to participate in complex regulatory networks via their regulation of transcription factors involved in water stress signaling. We explored the role of miR159 in the response of Solanum lycopersicum Mill. plants to drought stress by analyzing the expression of sly-miR159 and its target SlMYB transcription factor genes in tomato plants of cv. Ailsa Craig grown in deprived water conditions or in response to mechanical damage caused by the Colorado potato beetle, a devastating insect pest of Solanaceae plants. Results showed that sly-miR159 regulatory function in the tomato plants response to distinct stresses might be mediated by differential stress-specific MYB transcription factor targeting. sly-miR159 targeting of SlMYB33 transcription factor transcript correlated with accumulation of the osmoprotective compounds proline and putrescine, which promote drought tolerance. This highlights the potential role of sly-miR159 in tomato plants' adaptation to water deficit conditions.

Ammonium mediated changes in carbon and nitrogen metabolisms induce resistance against Pseudomonas syringae in tomato plants.

Predominant NH4+ nutrition causes an "ammonium syndrome" that induces metabolic changes and thereby provides resistance against Pseudomonas syringae infection through the activation of systemic acquired acclimation (SAA). Hence, to elucidate the mechanisms underlying NH4+-mediated SAA, the changes in nutrient balance and C and N skeletons were studied in NH4+-treated plants upon infection by P. syringae. A general decrease in cation and an increase in anion levels was observed in roots and leaves of NH4+-treated plants. Upon NH4+-based nutrition and infection, tomato leaves showed an accumulation of S, P, Zn, and of Mn. Mn accumulation might be required for ROS detoxification since it acts as a cofactor of superoxide dismutase (SOD). Primary metabolism was modified in both tissues of NH4+-fed plants to counteract NH4+ toxicity by decreasing TCA intermediates. A significant increase in Arg, Gln, Asn, Lys, Tyr, His and Leu was observed in leaves of NH4+-treated plants. The high level of the putrescine precursor Arg hints towards the importance of the Glu pathway as a key metabolic check-point in NH4+-treated and infected plants. Taken together, NH4+-fed plants displayed a high level of basal responses allowing them to activate SAA and to trigger defense responses against P. syringae through nutrient imbalances and changes in primary metabolism.

1-Methyltryptophan Modifies Apoplast Content in Tomato Plants Improving Resistance Against Pseudomonas syringae.

Plants can produce numerous natural products, many of which have been shown to confer protection against microbial attack. In this way, we identified 1-methyltryptophan (1-MT), a natural compound produced by tomato plants in response to Pseudomonas syringae attack, whose application by soil drench provided protection against this pathogen. In the present work, we have studied the mechanisms underlying this protection. The results demonstrated that 1-MT can be considered a new activator of plant defense responses that acts by inhibiting the stomatal opening produced by coronatine (COR) and could thereby, prevent bacteria entering the mesophyll. Besides, 1-MT acts by blocking the jasmonic acid (JA) pathway that, could avoid manipulation of the salicylic acid (SA) pathway by the bacterium, and thus hinder its growth. Although the concentration of 1-MT reached in the plant did not show antimicrobial effects, we cannot rule out a role for 1-MT acting alone because it affects the expression of the fliC gene that is involved in synthesis of the flagellum. These changes would result in reduced bacterium motility and, therefore, infective capacity. The results highlight the effect of a tryptophan derivative on induced resistance in plants.

Elucidating the mechanism of action of copper heptagluconate on the plant immune system against Pseudomonas syringae in tomato (Solanum lycopersicum L).

BACKGROUND: Phytopathogenic problems caused by the bacterial pathogen Pseudomonas syringae in tomato are becoming more serious due to the emergence of strains resistant to classical pesticides. This has led to research into new formulations with lower environmental problems. One of the most promising alternatives to the use of classical pesticides is the induction of natural plant defences. New formulations based on Cu complexed with heptagluconic acid induce plant innate defences and could be an alternative to classical treatments based on inorganic Cu against bacterial speck. To study the efficacy of this compound in tomato against P. syringae, we tested its systemic effect Applying the treatments via radicular. RESULTS: Treated plants showed less infection development and lower number of viable bacteria in leaves. We also observed better performance of parameters involved in plant resistance such as the antioxidant response and the accumulation of phenolic compounds. CONCLUSION: Results showed that soil drench applications can be highly effective for the prevention and control of bacterial speck in tomato plants, showing a reduction in symptoms of ∼ 50%. Moreover, application of Cu heptagluconate induced accumulation of the plant polyphenols caffeic and chlorogenic acids, and reduced the amount of reactive oxygen species in infected plants. © 2018 Society of Chemical Industry.

Comparative analysis of the immune responses induced by native versus recombinant versions of the ASP-based vaccine against the bovine intestinal parasite Cooperia oncophora.

The protective capacities of a native double-domain activation-associated secreted protein (ndd-ASP)-based vaccine against the cattle intestinal nematode Cooperia oncophora has previously been demonstrated. However, protection analysis upon vaccination with a recombinantly produced antigen has never been performed. Therefore, the aim of the current study was to test the protective potential of a Pichia-produced double-domain ASP (pdd-ASP)-based vaccine against C. oncophora. Additionally, we aimed to compare the cellular and humoral mechanisms underlying the vaccine-induced responses by the native (ndd-ASP) and recombinant vaccines. Immunisation of cattle with the native C. oncophora vaccine conferred significant levels of protection after an experimental challenge infection, whereas the recombinant vaccine did not. Moreover, vaccination with ndd-ASP resulted in a higher proliferation of CD4-T cells both systemically and in the small intestinal mucosa when compared with animals vaccinated with the recombinant antigen. In terms of humoral response, although both native and recombinant vaccines induced similar levels of antibodies, animals vaccinated with the native vaccine were able to raise antibodies with greater specificity towards ndd-ASP in comparison with antibodies raised by vaccination with the recombinant vaccine, suggesting a differential immune recognition towards the ndd-ASP and pdd-ASP. Finally, the observation that animals displaying antibodies with higher percentages of recognition towards ndd-ASP also exhibited the lowest egg counts suggests a potential relationship between antibody specificity and protection.

Epigenetic regulation of the expression of WRKY75 transcription factor in response to biotic and abiotic stresses in Solanaceae plants.

KEY MESSAGE: SlyWRKY75: gene expression was induced in response to biotic stresses, especially in Botrytis cinerea-infected tomato plants, in which Sly-miR1127-3p is a putative SlyWRKY75 regulator and epigenetic marks were detected. WRKY75 transcription factor involved in Pi homeostasis was recently found also induced in defense against necrotrophic pathogens. In this study, we analyzed by RT-qPCR the expression of SlyWRKY75 gene in tomato plants in response to abiotic stresses (drought or heat) and biotic stresses (Colorado potato beetle larvae infestation, Pseudomonas syringae or Botrytis cinerea infection) being only differentially expressed following biotic stresses, especially upon B. cinerea infection (55-fold induction). JA and JA-Ile levels were significantly increased in tomato plants under biotic stresses compared with control plants, indicating that SlyWRKY75 might be a transcriptional regulator of the JA pathway. The contribution of miRNAs and epigenetic molecular mechanisms to the regulation of this gene in B. cinerea-infected tomato plants was explored. We identified a putative Sly-miR1127-3p miRNA predicted to bind the intronic region of the SlyWRKY75 genomic sequence. Sly-miR1127-3p miRNA was repressed in infected plants (0.4-fold) supporting that it might act as an epigenetic regulation factor of SlyWRKY75 gene expression rather than via the post-transcriptional mechanisms of canonical miRNAs. It has been proposed that certain miRNAs can mediate DNA methylation in the plant nucleus broadening miRNA functions with transcriptional gene silencing by targeting intron-containing pre-mRNAs. Histone modifications analysis by chromatin immunoprecipitation (ChIP) demonstrated the presence of the activator histone modification H3K4me3 on SlyWRKY75 transcription start site and gene body. The induction of this gene in response to B. cinerea correlates with the presence of an activator mark. Thus, miRNAs and chromatin modifications might cooperate as epigenetic factors to modulate SlyWRKY75 gene expression.

Bioassimilable sulphur provides effective control of Oidium neolycopersici in tomato, enhancing the plant immune system.

BACKGROUND: Developments of alternatives to the use of chemical pesticides to control pests are focused on the induction of natural plant defences. The study of new compounds based on liquid bioassimilable sulphur and its effect as an inductor of the immune system of plants would provide an alternative option to farmers to enhance plant resistance against pathogen attacks such as powdery mildew. In order to elucidate the efficacy of this compound in tomato against powdery mildew, we tested several treatments: curative foliar, preventive foliar, preventive in soil drench and combining preventive in soil drench and curative foliar. RESULTS: In all cases, treated plants showed lower infection development, better physiological parameters and a higher level of chlorophyll. We also observed better performance in parameters involved in plant resistance such as antioxidant response, callose deposition and hormonal levels. CONCLUSION: The results indicate that preventive and curative treatments can be highly effective for the prevention and control of powdery mildew in tomato plants. Foliar treatments are able to stop the pathogen development when they are applied as curative. Soil drench treatments induce immune response mechanisms of plants, increasing significantly callose deposition and promoting plant development. © 2016 Society of Chemical Industry.

Host protective ASP-based vaccine against the parasitic nematode Ostertagia ostertagi triggers NK cell activation and mixed IgG1-IgG2 response.

The mucus-dwelling parasite Ostertagia ostertagi is one of the most important gastrointestinal nematodes in cattle. Our group has previously demonstrated the protective capacity of a vaccine against this parasite based on a native activation-associated secreted protein ASP1 (nASP) in combination with the saponin adjuvant QuilA. The aim of the current study was to analyse the effect of both antigen and adjuvant on the cellular and humoral vaccine-induced immune responses by comparing the native ASP to a recombinant version expressed in Pichia pastoris (pASP) and replacing QuilA by Al(OH)3. Immunization of cattle with the protective nASP+QuilA vaccine was associated with antigen-induced proliferation of natural killer (NK) cells combined with IFN-γ secretion and the induction of a mixed IgG1/IgG2 antibody response. ASP-specific activation and proliferation of NK cells was also observed in mice following the same vaccination regime. Replacing QuilA by Al(OH)3 or nASP by pASP significantly decreased the capacity of the vaccines to trigger both NK cell activation and antibody responses and failed to induce protection against a challenge infection. Reduction of the structurally anchoring disulphide bonds of the nASP completely abolished its ability to induce NK cell activation and antibody responses, highlighting the importance of protein conformation for the immunostimulatory activity.