Characterization of Biostimulant Mode of Action Using Novel Multi-Trait High-Throughput Screening of Arabidopsis Germination and Rosette Growth

Environmental stresses have a significant effect on agricultural crop productivity worldwide. Exposure of seeds to abiotic stresses, such as salinity among others, results in lower seed viability, reduced germination, and poor seedling establishment. Alternative agronomic practices, e.g., the use of plant biostimulants, have attracted considerable interest from the scientific community and commercial enterprises. Biostimulants, i.e., products of biological origin (including bacteria, fungi, seaweeds, higher plants, or animals) have significant potential for (i) improving physiological processes in plants and (ii) stimulating germination, growth and stress tolerance. However, biostimulants are diverse, and can range from single compounds to complex matrices with different groups of bioactive components that have only been partly characterized. Due to the complex mixtures of biologically active compounds present in biostimulants, efficient methods for characterizing their potential mode of action are needed. In this study, we report the development of a novel complex approach to biological activity testing, based on multi-trait high-throughput screening (MTHTS) of Arabidopsis characteristics. These include the in vitro germination rate, early seedling establishment capacity, growth capacity under stress and stress response. The method is suitable for identifying new biostimulants and characterizing their mode of action. Representatives of compatible solutes such as amino acids and polyamines known to be present in many of the biostimulant irrespective of their origin, i.e., well-established biostimulants that enhance stress tolerance and crop productivity, were used for the assay optimization and validation. The selected compounds were applied through seed priming over a broad concentration range and the effect was investigated simultaneously under control, moderate stress and severe salt stress conditions. The new MTHTS approach represents a powerful tool in the field of biostimulant research and development and offers direct classification of the biostimulants mode of action into three categories: (1) plant growth promotors/inhibitors, (2) stress alleviators, and (3) combined action

Arabidopsis plants lacking plastid phosphoglucose isomerase respond to microbial volatiles through GLUCOSE-6-P/PHOSPHATE TRANSLOCATOR2 action

Resumen del trabajo presentado en la XXIV Reunión de la Sociedad Española de Biología de Plantas - XVII Congreso Hispano-Luso de Biología de Plantas, celebrado online del 7 al 9 de julio de 2021Recent studies have shown that PGI1 is an important determinant of photosynthesis, growth and starch production, likely as a consequence of its involvement in the synthesis of plastidial isoprenoid compounds such as hormones and photosynthetic pigments (Bahaji et al. 2015; Bahaji et al. 2018). We have shown that volatile compounds (VCs) emitted by microbes promote growth, photosynthesis, and accumulation of cytokinins (CK) and starch (Sánchez-Lopez et al. 2016a). This response was PGI1-independent, as fungal VCs exposed PGI1 null pgi1-2 plants grew faster and accumulated exceedingly higher levels of starch and CKs than plants not exposed to VCs (Sánchez-López et al. 2016b). The most up-regulated gene in leaves of fungal VCs exposed plants was At1g61800, encoding the plastidial GPT2 glucose-6-phosphate (G6P)/phosphate translocator. Whether the response of pgi1-2 to fungal VCs could be due to the GPT2-mediated transport of cytosolic G6P into the chloroplast was investigated by characterizing pgi1-2/gpt2-2 PGI1- and GPT2-null double mutants cultured in the presence or absence of VCs emitted by the fungal phytopathogen Alternaria alternata. We found that photosynthesis, active CKs content, growth and leaf starch content in pgi1-2/gpt2-2 plants exposed to fungal VCs were lower than in VCs exposed WT, gpt2-2 and pgi1-2 plants. Proteomic analyses revealed that fungal VCs strongly up-regulate the expression of proteins involved in photosynthesis in WT, gpt2-2 and pgi1-2 plants, but in much lower extent in pgi1-2/gpt2-2 plants. The overall data show that the combined action of PGI1 and GTP2 is an important determinant of the plant´s response to microbial VCs. The possible involvement of these functions in the production of growth- and foliar metabolism-regulating isoprenoid hormones in heterotrophic organs is discussed.This work was supported by the Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (Spain) (grants BIO2016-78747-P and PID2019-104685GB-100) and the Ministry of Education, Youth and Sport of the Czech Republic and ERDF project “Plants as a tool for sustainable global development” (No. CZ.02.1.01/0.0/0.0/16_019/0000827).Peer reviewe

An Automated Method for High-Throughput Screening of Arabidopsis Rosette Growth in Multi-Well Plates and Its Validation in Stress Conditions

High-throughput plant phenotyping platforms provide new possibilities for automated, fast scoring of several plant growth and development traits, followed over time using non-invasive sensors. Using Arabidopsis as a model offers important advantages for high-throughput screening with the opportunity to extrapolate the results obtained to other crops of commercial interest. In this study we describe the development of a highly reproducible high-throughput Arabidopsis in vitro bioassay established using our OloPhen platform, suitable for analysis of rosette growth in multi-well plates. This method was successfully validated on example of multivariate analysis of Arabidopsis rosette growth in different salt concentrations and the interaction with varying nutritional composition of the growth medium. Several traits such as changes in the rosette area, relative growth rate, survival rate and homogeneity of the population are scored using fully automated RGB imaging and subsequent image analysis. The assay can be used for fast screening of the biological activity of chemical libraries, phenotypes of transgenic or recombinant inbred lines, or to search for potential quantitative trait loci. It is especially valuable for selecting genotypes or growth conditions that improve plant stress tolerance

Priming Treatments with Biostimulants to Cope the Short-Term Heat Stress Response: A Transcriptomic Profile Evaluation

Plant stress induced by high temperature is a problem in wide areas of different regions in the world. The trend of global warming is going to enhance the effects of heat stress on crops in many cultivation areas. Heat stress impairs the stability of cell membranes and many biological processes involving both primary and secondary metabolism. Biostimulants are innovative agronomical tools that can be used as a strategy to counteract the detrimental effect of abiotic stresses, including heat stress. In this work, two biostimulants based on Ascophyllum nodosum extracts (named Phylgreen) and based on animal L-α amino acids (named Delfan Plus) were applied as priming treatments to Arabidopsis thaliana plants subjected to heat stress exposure. Plants at the vegetative stage were treated with biostimulants 12 h before high temperature exposure, which consisted of maintaining the plants at 37 ± 1 °C for 4 h. Transcriptional profiles, physiological, and biochemical analyses were performed to understand the mode of action of the biostimulants in protecting the plants exposed to short-term heat stress. At a physiological level, chlorophyll, chlorophyll a fluorescence, phenolic index, total anthocyanins, reactive oxygen species (ROS) were measured, and significant variations were observed immediately after stress. Both biostimulants were able to reduce the oxidative damage in leaves and cell membrane. Transcriptomic data revealed that upregulated genes were 626 in Phylgreen and 365 in Delfan Plus, while downregulated genes were 295 in Phylgreen and 312 in Delfan Plus. Bioinformatic analysis showed that the biostimulants protected the plants from heat stress by activating specific heat shock proteins (HPS), antioxidant systems, and ROS scavengers. The results revealed that the biostimulants effectively induced the activation of heat stress-associated genes belonging to different transcription factors and HSP families. Among the heat shock proteins, the most important was the AtHSP17 family and in particular, those influenced by treatments were AtHPS17.4 and AtHPS17.6A, B, showing the most relevant changes

The combined action of plastidial phosphogklucose isomerase and glucose-6-P/Phosphate translocator2 is an important determinant of the response of plants to microbial volatiles

Trabajo presentado en la XIV Reunión de Biología Molecular de Plantas, celebrada en Salamanca (España) del 4 al 6 de julio de 2018Peer reviewe

Arabidopsis plants lacking plastid phosphoglucose isomerase respond to microbial volatiles through GLUCOSE-6-P/PHOSPHATE TRANSLOCATOR2 action

Resumen del trabajo presentado en la XV Reunión de Biología Molecular de Plantas, celebrada online los días 16 y 27 de noviembre de 2020Recent studies have shown that PGI1 is an important determinant of photosynthesis, growth and starch production, likely as a consequence of its involvement in the synthesis of plastidial isoprenoid compounds such as hormones and photosynthetic pigments. We have shown that volatile compounds (VCs) emitted by microbes promote growth,photosynthesis, and accumulation of cytokinins (CK) and starch. This response was PGI1-independent, as fungal VCs exposed PGI1 null pgi1-2 plants grew faster and accumulated exceedingly higher levels of starch and CKs than plants not exposed to VCs. The most up-regulated gene in leaves of fungal VCs exposed plants was At1g61800, encoding the plastidial GPT2 glucose-6-phosphate (G6P)/phosphate translocator. Whether the response of pgi1-2 to fungal VCs could be due to the GPT2-mediated transport of cytosolic G6P into the chloroplast was investigated by characterizing pgi1-2/gpt2-2 PGI1- and GPT2-null double mutants cultured in the presence or absence of VCs emitted by the fungal phytopathogen Alternaria alternata. We found that photosynthesis, active CK content, growth and leaf starch content in pgi1-2/gpt2-2 plants exposed to fungal VCs were lower than in VCs exposed WT, gpt2-2 and pgi1-2 plants. Proteomic analyses revealed that fungal VCs strongly up-regulate the expression of proteins involved in photosynthesis in WT, gpt2-2 and pgi1-2 plants, but in much lower extent in pgi1-2/gpt2-2 plants. The overall data show that the combined action of PGI1 and GTP2 is an important determinant of the plant¿s response to microbial VCs. The possible involvement of these functions in the production of growthand foliar metabolism-regulating isoprenoid hormones in heterotrophic organs is discussed.This work was supported by the Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (Spain) (grants BIO2016-78747-P and PID2019-104685GB100) and the Ministry of Education, Youth and Sport of the Czech Republic and ERDF project “Plants as a tool for sustainable global development” (No. CZ.02.1.01/0.0/0.0/16_019/0000827).Peer reviewe

Volatiles from the fungal phytopathogen Penicillium aurantiogriseum modulate root metabolism and architecture through proteome resetting

Volatile compounds (VCs) emitted by the fungal phytopathogen Penicillium aurantiogriseum promote root growth and developmental changes in Arabidopsis. Here we characterized the metabolic and molecular responses of roots to fungal volatiles. Proteomic analyses revealed that these compounds reduce the levels of aquaporins, the iron carrier IRT1 and apoplastic peroxidases. Fungal VCs also increased the levels of enzymes involved in the production of mevalonate (MVA)‐derived isoprenoids, nitrogen assimilation and conversion of methionine to ethylene and cyanide. Consistently, fungal VC‐treated roots accumulated high levels of hydrogen peroxide (H2O2), MVA‐derived cytokinins, ethylene, cyanide and long‐distance nitrogen transport amino acids. qRT‐PCR analyses showed that many proteins differentially expressed by fungal VCs are encoded by VC non‐responsive genes. Expression patterns of hormone reporters and developmental characterization of mutants provided evidence for the involvement of cyanide scavenging and enhanced auxin, ethylene, cytokinin and H2O2 signaling in the root architecture changes promoted by fungal VCs. Our findings show that VCs from P. aurantiogriseum modify root metabolism and architecture, and improve nutrient and water use efficiencies through transcriptionally and non‐transcriptionally regulated proteome resetting mechanisms. Some of these mechanisms are subject to long‐distance regulation by photosynthesis and differ from those triggered by VCs emitted by beneficial microorganisms.Peer reviewe

Plant responses to fungal volatiles involve global posttranslational thiol redox proteome changes that affect photosynthesis

Microorganisms produce volatile compounds (VCs) that promote plant growth and photosynthesis through complex mechanisms involving cytokinin (CK) and abscisic acid (ABA). We hypothesized that plants' responses to microbial VCs involve posttranslational modifications of the thiol redox proteome through action of plastidial NADPH‐dependent thioredoxin reductase C (NTRC), which regulates chloroplast redox status via its functional relationship with 2‐Cys peroxiredoxins. To test this hypothesis, we analysed developmental, metabolic, hormonal, genetic, and redox proteomic responses of wild‐type (WT) plants and a NTRC knockout mutant (ntrc) to VCs emitted by the phytopathogen Alternaria alternata. Fungal VC‐promoted growth, changes in root architecture, shifts in expression of VC‐responsive CK‐ and ABA‐regulated genes, and increases in photosynthetic capacity were substantially weaker in ntrc plants than in WT plants. As in WT plants, fungal VCs strongly promoted growth, chlorophyll accumulation, and photosynthesis in ntrc–Δ2cp plants with reduced 2‐Cys peroxiredoxin expression. OxiTRAQ‐based quantitative and site‐specific redox proteomic analyses revealed that VCs promote global reduction of the thiol redox proteome (especially of photosynthesis‐related proteins) of WT leaves but its oxidation in ntrc leaves. Our findings show that NTRC is an important mediator of plant responses to microbial VCs through mechanisms involving global thiol redox proteome changes that affect photosynthesis.This work was supported by the Comisión Interministerial de Ciencia y Tecnología and Fondo Europeo de Desarrollo Regional (Spain; Grants BIO2013‐49125‐C2‐1‐P, BIO2016‐78747‐P, and BIO2017‐85195‐C2‐1‐P), the Government of Navarra (Refs. P1044 AGROESTI and P1004 PROMEBIO), the KAKENHI Grants‐in‐Aid for Scientific Research (A; 15H02486) from the Japan Society for the Promotion of Sciences, Strategic International Collaborative Research Program by the Japan Science and Technology Agency (JST SICORP), the Grant for Promotion of KAAB Projects (Niigata University) from the Ministry of Education, Culture, Sports, Science and Technology (Japan) and the Ministry of Education, Youth and Sport of the Czech Republic, and European Regional Development Fund project “Plants as a tool for sustainable global development” (CZ.02.1.01/0.0/0.0/16_019/0000827).Peer reviewe