Humic Substances Isolated from Recycled Biomass Trigger Jasmonic Acid Biosynthesis and Signalling.

Intensive agriculture maintains high crop yields through chemical inputs, which are well known for their adverse effects on environmental quality and human health. Innovative technologies are required to reduce the risk generated by the extensive and harmful use of pesticides. The plant biostimulants made from humic substances isolated from recyclable biomass offer an alternative approach to address the need for replacing conventional agrochemicals without compromising the crop yield. The stimulatory effects of humic substances are commonly associated with plant hormones, particularly auxins. However, jasmonic acid (JA) is crucial metabolite in mediating the defence responses and governing plant growth and development. This work aimed to evaluate the changes in the biosynthesis and signalling pathway of JA in tomato seedlings treated with humic acids (HA) isolated from vermicompost. We use the tomato model system cultivar Micro-Tom (MT) harbouring a reporter gene fused to a synthetic promoter that responds to jasmonic acid (JERE::GUS). The transcript levels of genes involved in JA generation and activity were also determined using qRT-PCR. The application of HA promoted plant growth and altered the JA status, as revealed by both GUS and qRT-PCR assays. Both JA enzymatic synthesis (LOX, OPR3) and JA signalling genes (JAZ and JAR) were found in higher transcription levels in plants treated with HA. In addition, ethylene (ETR4) and auxin (ARF6) signalling components were positively modulated by HA, revealing a hormonal cross-talk. Our results prove that the plant defence system linked to JA can be emulated by HA application without growth inhibition.

Changes in metabolic profiling of sugarcane leaves induced by endophytic diazotrophic bacteria and humic acids.

Plant growth-promoting bacteria (PGPB) and humic acids (HA) have been used as biostimulants in field conditions. The complete genomic and proteomic transcription of Herbaspirillum seropedicae and Gluconacetobacter diazotrophicus is available but interpreting and utilizing this information in the field to increase crop performance is challenging. The identification and characterization of metabolites that are induced by genomic changes may be used to improve plant responses to inoculation. The objective of this study was to describe changes in sugarcane metabolic profile that occur when HA and PGPB are used as biostimulants. Inoculum was applied to soil containing 45-day old sugarcane stalks. One week after inoculation, the methanolic extracts from leaves were obtained and analyzed by gas chromatography coupled to time-of-flight mass spectrometry; a total of 1,880 compounds were observed and 280 were identified in all samples. The application of HA significantly decreased the concentration of 15 metabolites, which generally included amino acids. HA increased the levels of 40 compounds, and these included metabolites linked to the stress response (shikimic, caffeic, hydroxycinnamic acids, putrescine, behenic acid, quinoline xylulose, galactose, lactose proline, oxyproline and valeric acid) and cellular growth (adenine and adenosine derivatives, ribose, ribonic acid and citric acid). Similarly, PGPB enhanced the level of metabolites identified in HA-treated soils; e.g., 48 metabolites were elevated and included amino acids, nucleic acids, organic acids, and lipids. Co-inoculation (HA+PGPB) boosted the level of 110 metabolites with respect to non-inoculated controls; these included amino acids, lipids and nitrogenous compounds. Changes in the metabolic profile induced by HA+PGPB influenced both glucose and pentose pathways and resulted in the accumulation of heptuloses and riboses, which are substrates in the nucleoside biosynthesis and shikimic acid pathways. The mevalonate pathway was also activated, thus increasing phytosterol synthesis. The improvement in cellular metabolism observed with PGPB+HA was compatible with high levels of vitamins. Glucuronate and amino sugars were stimulated in addition to the products and intermediary compounds of tricarboxylic acid metabolism. Lipids and amino acids were the main compounds induced by co-inoculation in addition to antioxidants, stress-related metabolites, and compounds involved in cellular redox. The primary compounds observed in each treatment were identified, and the effect of co-inoculation (HA+PGPB) on metabolite levels was discussed.

Changes in labile phosphorus forms during maturation of vermicompost enriched with phosphorus-solubilizing and diazotrophic bacteria.

The aim of this study was to assess the effect of N(2)-fixing and P-solubilizing bacteria during maturation of vermicompost on phosphorus availability. A bacterial suspension containing Burkholderia silvatlantica, Burkholderia spp. and Herbaspirillum seropedicae was applied at the initial stage of vermicomposting. At the end of the incubation time (120days), the nitrogen content had increased by18% compared to uninoculated vermicompost. Water-soluble P was 106% higher in inoculated vermicompost while resin-extractable P increased during the initial vermicomposting stage and was 21% higher at 60days, but was the same in inoculated and uninoculated mature compost. The activity of acid phosphatase was 43% higher in inoculated than uninoculated vermicompost. These data suggest that the introduction of the mixed culture had beneficial effects on vermicompost maturation.

Nitric oxide mediates humic acids-induced root development and plasma membrane H+-ATPase activation.

It is widely reported that some humic substances behave as exogenous auxins influencing root growth by mechanisms that are not yet completely understood. This study explores the hypothesis that the humic acids' effects on root development involve a nitric oxide signaling. Maize seedlings were treated with HA 20 mg C L(-1), IAA 0.1 nM, and NO donors (SNP or GSNO), in combination with either the auxin-signaling inhibitor PCIB, the auxin efflux inhibitor TIBA, or the NO scavenger PTIO. H(+)-transport-competent plasma membrane vesicles were isolated from roots to investigate a possible link between NO-induced H(+)-pump and HA bioactivity. Plants treated with either HA or SNP stimulated similarly the lateral roots emergence even in the presence of the auxin inhibitors, whereas NO scavenger diminished this effect. These treatments induced H(+)-ATPase stimulation by threefold, which was abolished by PTIO and decreased by auxin inhibitors. HA-induced NO synthesis was also detected in the sites of lateral roots emergence. These data depict a new scenario where the root development stimulation and the H(+)-ATPase activation elicited by either HA or exogenous IAA depend essentially on mechanisms that use NO as a messenger induced site-specifically in the early stages of lateral root development.

Chemical composition and bioactivity properties of size-fractions separated from a vermicompost humic acid.

Preparative high performance size-exclusion chromatography (HPSEC) was applied to humic acids (HA) extracted from vermicompost in order to separate humic matter of different molecular dimension and evaluate the relationship between chemical properties of size-fractions (SF) and their effects on plant root growth. Molecular dimensions of components in humic SF was further achieved by diffusion-ordered nuclear magnetic resonance spectroscopy (DOSY-NMR) based on diffusion coefficients (D), while carbon distribution was evaluated by solid state (CP/MAS) (13)C NMR. Seedlings of maize and Arabidopsis were treated with different concentrations of SF to evaluate root growth. Six different SF were obtained and their carbohydrate-like content and alkyl chain length decreased with decreasing molecular size. Progressive reduction of aromatic carbon was also observed with decreasing molecular size of separated fractions. Diffusion-ordered spectroscopy (DOSY) spectra showed that SF were composed of complex mixtures of aliphatic, aromatic and carbohydrates constituents that could be separated on the basis of their diffusion. All SF promoted root growth in Arabidopsis and maize seedlings but the effects differed according to molecular size and plant species. In Arabidopsis seedlings, the bulk HA and its SF revealed a classical large auxin-like exogenous response, i.e.: shortened the principal root axis and induced lateral roots, while the effects in maize corresponded to low auxin-like levels, as suggested by enhanced principal axis length and induction of lateral roots. The reduction of humic heterogeneity obtained in HPSEC separated size-fractions suggested that their physiological influence on root growth and architecture was less an effect of their size than their content of specific bioactive molecules. However, these molecules may be dynamically released from humic superstructures and exert their bioactivity when weaker is the humic conformational stability as that obtained in the separated size-fractions.