A Fast and Precise Method To Identify Indolic Glucosinolates and Camalexin in Plants by Combining Mass Spectrometric and Biological Information

In this manuscript, a fast and accurate identification and quantitation by mass spectrometry of indolic glucosinolates and camalexin involved in defense in Arabidopsis thaliana are described. Two elicitation systems, inoculation with Botrytis cinerea and treatment with AgNO3, were used in Col-0 wild-type and mutant genotypes impaired in the biosynthesis of the selected metabolites. Identification of analytes was carried out by nontargeted LC/ESI-QTOF-MS profiling. Confirmation of indolic glucosinolates and camalexin was achieved by their absence in the cyp79B2/B3 and pad3 mutants as well as their respective fragmentation upon collision-induced dissociation. Camalexin accumulation was induced only after AgNO3 treatment, whereas all indolic glucosinolates were constitutively present. Inoculation with Botrytis did not influence camalexin concentration but caused most aliphatic and indolic glucosinolates contents to decrease. Only the pen 3.1 mutant showed increased indolic glucosinolate levels after Botrytis or AgNO3 treatments. In addition, profiles of secondary metabolite in nontreated Col-0 and mutant plants were analyzed by means of partial least squares coupled to discriminant analysis (PLS-DA), and differences in the basal levels of indolic glucosinolates and tryptophan between cyp79B2/B3 plants and the rest of genotypes, including Col-0, were found. This probably has to be taken into consideration when comparing stress responses of Col-0 and cyp79B2/B3. The use of mutants carrying alterations in biosynthetic pathways is proposed as a useful strategy to identify secondary metabolites

Metabolomics of Disease Resistance in Crops

Plants are continuously exposed to the attack of invasive microorganisms, such as fungi or bacteria, and also viruses. To fight these attackers, plants develop different metabolic and genetic responses whose final outcome is the production of toxic compounds that kill the pathogen or deter its growth or semiotic molecules that alert other individuals of the same plant species. These molecules are derived from the secondary metabolism and their production is induced upon detection of a pathogen-associated molecular pattern (PAMP). These PAMPs are different molecules that are perceived by the host cell triggering defense responses. PAMP-elicited compounds are highly diverse and specific of every plant species and can be divided into preformed metabolites or phytoanticipins that are converted into toxic molecules upon pathogen perception, and toxic metabolites or phytoalexins that are produced only upon pathogen attack. Moreover, plant volatile emissions are also modified in response to pathogen attack to alert neighboring individuals or to make plants less attractive to pathogen vector arthropods. Plant metabolite profiling techniques have allowed the identification of novel antimicrobial molecules that are induced upon elicitation. However, more studies are required to assess the specific function of metabolites or metabolite blends on plant-microbe interactions

Metabolic and Regulatory Responses in Citrus rootstock in Response to Adverse Environmental Conditions

In response to adverse environmental conditions, plants modify their metabolism to adapt to the new conditions. To differentiate common responses to abiotic stress from specific adaptation to a certain stress condition, two citrus rootstocks (Carrizo citrange and Cleopatra mandarin) with a different ability to tolerate stress were subjected to soil flooding and drought, two water stress conditions. In response to these conditions, both genotypes showed altered root proline and phenylpropanoid levels, especially cinnamic acid, which was a common feature to Carrizo and Cleopatra. This was correlated with alterations in the levels of phenylpropanoid derivatives likely involved in lignin biosynthesis. In the regulatory part, levels of both stress hormones abscisic acid (ABA) and jasmonic acid (JA) decreased in response to soil flooding irrespective of the genotype’s relative flooding tolerance, but, on the other hand, the concentration of both metabolites increased in response to drought, showing a transient accumulation of JA after a few days and a progressive pattern of ABA increase. These responses are probably associated with different regulatory processes under soil flooding and drought. In addition, alterations in indole acetic acid (IAA) levels in citrus roots seemed to be associated with particular stress tolerance. Moreover, both genotypes exhibited a low degree of overlap in the metabolites induced under similar stress conditions, indicating a specific mechanism to cope with stress in plant species. Results also indicated a different metabolic basal status in both genotypes that could contribute to stress tolerance

Abscisic Acid as an Emerging Modulator of the Responses of Plants to Low Oxygen Conditions

Different environmental and developmental cues involve low oxygen conditions, particularly those associated to abiotic stress conditions. It is widely accepted that plant responses to low oxygen conditions are mainly regulated by ethylene (ET). However, interaction with other hormonal signaling pathways as gibberellins (GAs), auxin (IAA), or nitric oxide (NO) has been well-documented. In this network of interactions, abscisic acid (ABA) has always been present and regarded to as a negative regulator of the development of morphological adaptations to soil flooding: hyponastic growth, adventitious root emergence, or formation of secondary aerenchyma in different plant species. However, recent evidence points toward a positive role of this plant hormone on the modulation of plant responses to hypoxia and, more importantly, on the ability to recover during the post-hypoxic period. In this work, the involvement of ABA as an emerging regulator of plant responses to low oxygen conditions alone or in interaction with other hormones is reviewed and discussed

Salt stress alleviation in citrus plants by plant growth-promoting rhizobacteria Pseudomonas putida and Novosphingobium sp.

Key message This work reveals the protective role of two rhizobacteria, Pseudomonas putida and Novosphingobium sp., on citrus plants subjected to salt stress conditions. Abstract Detrimental salt stress effects on crops are likely to increase due to climate change reducing the quality of irrigation water. Plant growth-promoting rhizobacteria (PGPRs) can mitigate stress-induced damage in plants cultivated under high salinity conditions. In this work, Citrus macrophylla (alemow) plants inoculated with the rhizobacteria Pseudomonas putida KT2440 or Novosphingobium sp. HR1a were subjected to salt stress for 30 days. Results showed that in absence of salt stress, Novosphingobium sp. HR1a induced a decrease of transpiration (E) and stomatal conductance (gs). Both rhizobacteria reduced salt stress-induced damage. Levels of abscisic acid (ABA) and salicylic acid (SA) were lower in inoculated plants under salt stress conditions. Similarly, under stress conditions maximum efficiency of photosystem II (Fv/Fm) in inoculated plants decreased to a lower extent than in non-inoculated ones. In stressed plants, Novosphingobium sp. HR1a also induced leaf accumulation of 3-indole acetic acid (IAA) and a delay in the decrease of quantum yield (ΦPSII). P. putida KT2440 inhibited root chloride and proline accumulation in response to salt stress. Although both bacterial species had beneficial effects on salt-stressed citrus plants, Novosphingobium sp. HR1a induced a better plant performance. Therefore, both strains could be candidates to be used as PGPRs in programs of inoculation for citrus protection against salt stress

Rapid and reproducible determination of active gibberellins in citrus tissues by UPLC/ESI-MS/MS

Phytohormone determination is crucial to explain the physiological mechanisms during growth and development. Therefore, rapid and precise methods are needed to achieve reproducible determination of phytohormones. Among many others, gibberellins (GAs) constitute a family of complex analytes as most of them share similar structure and chemical properties although only a few hold biological activity (namely GA1; GA3; GA4 and GA7). A method has been developed to extract GAs from plant tissues by mechanical disruption using ultrapure water as solvent and, in this way, ion suppression was reduced whereas sensitivity increased. Using this methodology, the four active GAs were separated and quantified by UPLC coupled to MS/MS using the isotope-labeled internal standards [2 H2]-GA1 and [2 H2]-GA4. To sum up, the new method provides a fast and reproducible protocol to determine bioactive GAs at low concentrations, using minimal amounts of sample and reducing the use of organic solvents.Ministerio de Economia (MINECO) AGL2013-42038-R Universitat Jaume I P1IB2013-23 "Santiago Grisolia" grant from Generalitat Valenciana "Ramon y Cajal" contract from MINEC

Arabidopsis thaliana DOF6 negatively affects germination in non-after ripened seeds and interacts with TCP14

Seed dormancy prevents seeds from germinating under environmental conditions unfavourable for plant growth and development and constitutes an evolutionary advantage. Dry storage, also known as after-ripening, gradually decreases seed dormancy by mechanisms not well understood. An Arabidopsis thaliana DOF transcription factor gene (DOF6) affecting seed germination has been characterized. The transcript levels of this gene accumulate in dry seeds and decay gradually during after-ripening and also upon seed imbibition. While constitutive over-expression of DOF6 produced aberrant growth and sterility in the plant, its over-expression induced upon seed imbibition triggered delayed germination, abscisic acid (ABA)-hypersensitive phenotypes and increased expression of the ABA biosynthetic gene ABA1 and ABA-related stress genes. Wild-type germination and gene expression were gradually restored during seed after-ripening, despite of DOF6-induced over-expression. DOF6 was found to interact in a yeast two-hybrid system andin planta with TCP14, a previously described positive regulator of seed germination. The expression of ABA1 and ABA-related stress genes was also enhanced in tcp14 knock-out mutants. Taken together, these results indicate that DOF6 negatively affects seed germination and opposes TCP14 function in the regulation of a specific set of ABA-related gene

NaCl protects against Cd and Cu-induced toxicity in the halophyte Atriplex halimus

The objective of the present work was to evaluate the extent of Cd- and Cu-induced oxidative stress and the antioxidant response triggered in the halophyte species Atriplex halimus after metallic trace elements exposure. Plants were treated for one month with Cd2+ or Cu2+ (400 μM) in the absence or presence of 200 mM NaCl in the irrigation solution. The interaction between salinity and heavy metal stress was analyzed in relation to plant growth, tissue ion contents (Na+, K+ and Mg2+), oxidative damage and antioxidative metabolism. Data indicate that shoot and root weight significantly decreased as a consequence of Cd2+- or Cu2+-induced stress. Metallic stress leads to unbalanced nutrient uptake by reducing the translocation of K+ and Mg2+ from the root to the shoot. The levels of malondialdehyde increased in root tissue when Cd, and especially Cu, were added to the irrigation solution, indicating that oxidative damage occurred. Results showed that NaCl gave a partial protection against Cd and Cu induced toxicity, although these contaminants had distinct influence on plant physiology. It can be concluded that salinity drastically modified heavy metal absorption and improved plant growth. Salinity also decreased oxidative damage, but differently in plants exposed to Cd or Cu stress.Ministerio de Economía y Competitividad, Spain (GL2013-42038-R; AGL2016-76574-R); Universitat Jaume I (P1- 1B2012-06)

Grafting improves tolerance to combined drought and heat stresses by modifying metabolism in citrus scion

Mediterranean basin and other citrus-growing areas, such as Florida or California, are among the most vulnerable regions to the impacts of global warming. Therefore, citrus will be likely subjected to increasing periods of water scarcity combined with high temperatures that will impair plant growth, development and yield. In citrus industry, grafting is used to improve varietal characteristics, such as abiotic stress tolerance. Previous research showed that under drought and heat stress conditions, Carrizo citrange (Poncirus trifoliata × Citrus sinensis) is a better rootstock than Cleopatra mandarin (Citrus reshni) because it induces a higher antioxidant activity on the scion, reducing oxidative damage and increasing plant tolerance. Here, it is shown that metabolic reconfiguration, including changes in carbohydrate and amino acid fluxes, are key responses for plant acclimation to stress conditions. Moreover, the importance of the rootstock on scion metabolic and hormonal responses to drought and heat stress combination has been addressed by using reciprocal grafting between Carrizo and Cleopatra genotypes. Thus, Carrizo as a rootstock improves the metabolic and hormonal response of Cleopatra scions to the stress combination by inducing the accumulation of protective compounds such as raffinose, galactinol and salicylic acid. In turn, Cleopatra as a rootstock reduces levels of raffinose, galactinol, proline, phenylalanine and tryptophan in Carrizo scions, which impairs plant tolerance to the stress combination. Our findings show the effect of the rootstock on scion metabolic response to stress combination and remark the importance of the rootstock in citrus plants exposed to harsh environmental conditions.Funding for open access charge: CRUE-Universitat Jaume

Biochemical and hormonal changes associated with root growth restriction under cadmium stress during maize (Zea mays L.) pre-emergence

Cadmium (Cd) pollution of agricultural soils is a growing global concern. Plant growth restriction is the main visible symptom of Cd toxicity, and this metal may be particularly harmful to the preformed, seminal root during the pre-emergence stage. In the present study, we focused on Cd phytotoxicity in seminal root growth, nutrient composition, redox status, and hormone homeostasis during the pre-emergence stage of maize (Zea mays L) plants, distinguishing between the root apex and the remaining root tissue. After 72 h of metal exposure (50 and 100 µM CdCl2), root length and biomass, as well as Ca, Fe, Mg, and Mn contents, were diminished. A redox imbalance was evidenced by changes in peroxidase activities and the ascorbate–dehydroascorbate ratio decreased in both root parts. There were fewer carbonylated proteins in both root fractions after exposure to 50 µM Cd, compared to 100 µM Cd, which was related to increased 20S proteasome activities. Cd incremented ABA, IAA, and SA contents, but drastically reduced the biologically active gibberellin GA4 and the conjugate jasmonoyl-isoleucine (JA-Ile). We demonstrated that the whole root tissue is involved in the maize response to Cd stress, which entails redox and hormonal rearrangements, probably directed to widen the plant defense lines at the expense of root growth