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.

Department of Chemical Biology and Genetics Centre of the Region Haná for Biotechnological and Agricultural Research Faculty of Science Palacký University Olomouc Czechia

Laboratory of Growth Regulators Centre of the Region Haná for Biotechnological and Agricultural Research Institute of Experimental Botany Czech Academy of Sciences Olomouc Czechia

Zobrazit více v PubMed

Calvo P., Nelson L., Kloepper J. W. (2014). Agricultural uses of plant biostimulants. Plant Soil 383 3–41. 10.1007/s11104-014-2131-8 DOI

Chen K., Arora R. (2013). Priming memory invokes seed stress-tolerance. Environ. Exp. Bot. 94 33–45. 10.1016/j.envexpbot.2012.03.005 PubMed DOI

Colla G., Rouphael Y., Canaguier R., Svecova E., Cardarelli M. (2014). Biostimulant action of a plant-derived protein hydrolysate produced through enzymatic hydrolysis. Front. Microbiol. 5:448. 10.3389/fpls.2014.00448 PubMed DOI PMC

Craigie J. S. (2011). Seaweed extract stimuli in plant science and agriculture. J. Appl. Phycol. 23 371–393. 10.1007/s10811-010-9560-4 DOI

Cristiano G., Pallozzi E., Conversa G., Tufarelli V., De Lucia B. (2018). Effects of an animal-derived biostimulant on the growth and physiological parameters of potted snapdragon (Antirrhinum majus L.). Front. Plant Sci. 9:861. 10.3389/fpls.2018.00861 PubMed DOI PMC

Dawood M. G., Taie H. A. A., Nassar R. M. A., Abdelhamid M. T., Schmidhalter U. (2014). The changes induced in the physiological, biochemical and anatomical characteristics of Vicia faba by the exogenous application of proline under seawater stress. South African J. Bot. 93 54–63. 10.1016/j.sajb.2014.03.002 DOI

De Diego N., Fürst T., Humplík J. F., Ugena L., Podlešáková K., Spíchal L. (2017). An automated method for high-throughput screening of Arabidopsis rosette growth in multi-well plates and its validation in stress conditions. Front. Plant Sci. 8:1702. 10.3389/fpls.2017.01702 PubMed DOI PMC

De Diego N., Saiz-Fernandez I., Rodriguez J. L., Perez-Alfocea F., Sampedro M. C., Barrio R. J., et al. (2015). Metabolites and hormones are involved in the intraspecific variability of drought hardening in radiata pine. J. Plant Physiol. 188 64–71. 10.1016/j.jplph.2015.08.006 PubMed DOI

Deivanai S., Xavier R., Vinod V., Timalata K., Lim O. F. (2011). Role of exogenous proline in ameliorating salt stress at early stage in two rice cultivars. J. Stress Physiol. Biochem. 7 157–174.

du Jardin P. (2015). Plant biostimulants: definition, concept, main categories and regulation. Sci. Hortic. 196 3–14. 10.1016/j.scienta.2015.09.021 DOI

Durand N., Briand X., Meyer C. (2003). The effect of marine bioactive substances (N PRO) and exogenous cytokinins on nitrate reductase activity in Arabidopsis thaliana. Physiol. Plant. 119 489–493. 10.1046/j.1399-3054.2003.00207.x DOI

Faragó D., Sass L., Valkai I., Andrási N., Szabados L. (2018). PlantSize offers an affordable, non-destructive method to measure plant size and color in vitro. Front. Plant Sci. 9:219. 10.3389/fpls.2018.00219 PubMed DOI PMC

Garcia-Gonzalez J., Sommerfeld M. (2016). Biofertilizer and biostimulant properties of the microalga Acutodesmus dimorphus. J. Appl. Phycol. 28 1051–1061. 10.1007/s10811-015-0625-2 PubMed DOI PMC

Gitelson A. A., Kaufman Y. J., Stark R., Rundquist D. (2002). Novel algorithms for remote estimation of vegetation fraction. Remote Sens. Environ. 80 76–87. 10.1016/S0034-4257(01)00289-9 DOI

Granier C., Granier C., Aguirrezabal L., Aguirrezabal L., Chenu K., Chenu K., et al. (2006). PHENOPSIS, an automated platform for reproducible phenotyping of plant responses to soil water de cit in. New Phytol. 169 623–635. 10.1111/j.1469-8137.2005.01609.x PubMed DOI

Hunt E. R., Doraiswamy P. C., McMurtrey J. E., Daughtry C. S. T., Perry E. M., Akhmedov B. (2013). A visible band index for remote sensing leaf chlorophyll content at the canopy scale. Int. J. Appl. Earth Obs. Geoinf. 21 103–112. 10.1016/J.JAG.2012.07.020 DOI

Ibrahim E. A. (2016). Seed priming to alleviate salinity stress in germinating seeds. J. Plant Physiol. 192 38–46. 10.1016/j.jplph.2015.12.011 PubMed DOI

Kaveh H., Nemati H., Farsi I. M., Vatandoost Jartoodeh S. (2011). How salinity affect germination and emergence of tomato lines. J. Biol. Environ. Sci. 5 159–163.

Li J., Hu L., Zhang L., Pan X., Hu X. (2015). Exogenous spermidine is enhancing tomato tolerance to salinity-alkalinity stress by regulating chloroplast antioxidant system and chlorophyll metabolism. BMC Plant Biol. 15:303. 10.1186/s12870-015-0699-7 PubMed DOI PMC

Mahdavi B. (2013). Seed germination and growth responses of Isabgol (Plantago ovata Forsk) to chitosan and salinity. Int. J. Agric. Crop Sci. 5 1084–1088.

Medeiros M. J. L., Silva M. M. A., Granja M. M. C., Souza e Silva Júnior G., Camara T. R., Willadino L., et al. (2015). Effect of exogenous proline in two sugarcane genotypes grown in vitro under salt stress. Acta biol. Colomb. 20 57–63. 10.15446/abc.v20n2.42830 DOI

Murashige T., Skoog F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 15 473–497. 10.1111/j.1399-3054.1962.tb08052.x DOI

Perry E. M., Roberts D. A. (2008). Sensitivity of narrow-band and broad-band indices for assessing nitrogen availability and water stress in an annual crop. Agron. J. 100:1211 10.2134/agronj2007.0306 DOI

Pichyangkura R., Chadchawan S. (2015). Biostimulant activity of chitosan in horticulture. Sci. Hortic. 196 49–65. 10.1016/j.scienta.2015.09.031 DOI

Podlešáková K., Ugena L., Spíchal L., Doležal K., De Diego N. (2018). Phytohormones and polyamines regulate plant stress responses by altering GABA pathway. N. Biotechnol. 10.1016/j.nbt.2018.07.003 [Epub ahead of print]. PubMed DOI

Pouvreau J.-B., Gaudin Z., Auger B., Lechat M.-M., Gauthier M., Delavault P., et al. (2013). A high-throughput seed germination assay for root parasitic plants. Plant Methods 9:32. 10.1186/1746-4811-9-32 PubMed DOI PMC

Povero G., Mejia J. F., Di Tommaso D., Piaggesi A., Warrior P. (2016). A systematic approach to discover and characterize natural plant biostimulants. Front. Plant Sci. 7:435. 10.3389/fpls.2016.00435 PubMed DOI PMC

Rahaman M. M., Ahsan M. A., Gillani Z., Chen M. (2017). Digital biomass accumulation using high-throughput plant phenotype data analysis. J. Integr. Bioinform. 14 1–13. 10.1515/jib-2017-0028 PubMed DOI PMC

Rodriguez-Furlán C., Miranda G., Reggiardo M., Hicks G. R., Norambuena L. (2016). High throughput selection of novel plant growth regulators: assessing the translatability of small bioactive molecules from Arabidopsis to crops. Plant Sci. 245 50–60. 10.1016/j.plantsci.2016.01.001 PubMed DOI

Roychoudhury A., Basu S., Sengupta D. N. (2011). Amelioration of salinity stress by exogenously applied spermidine or spermine in three varieties of indica rice differing in their level of salt tolerance. J. Plant Physiol. 168 317–328. 10.1016/j.jplph.2010.07.009 PubMed DOI

Sabagh A., El Islam M. S., Ueda A., Saneoka H., Barutçular C. (2015). Increasing reproductive stage tolerance to salinity stress in soybean. Int. J. Agric. Crop Sci. 8 738–745.

Savvides A., Ali S., Tester M., Fotopoulos V. (2016). Chemical priming of plants against multiple abiotic stresses: mission possible? Trends Plant Sci. 21 329–340. 10.1016/j.tplants.2015.11.003 PubMed DOI

Sharma H. S. S., Fleming C., Selby C., Rao J. R., Martin T. (2014). Plant biostimulants: a review on the processing of macroalgae and use of extracts for crop management to reduce abiotic and biotic stresses. J. Appl. Phycol. 26 465–490. 10.1007/s10811-013-0101-9 DOI

Sharma H. S. S., Selby C., Carmichael E., McRoberts C., Rao J. R., Ambrosino P., et al. (2016). Physicochemical analyses of plant biostimulant formulations and characterisation of commercial products by instrumental techniques. Chem. Biol. Technol. Agric. 3:13 10.1186/s40538-016-0064-6 DOI

Shekari F., Danalo A. A., Mustafavi S. H. (2015). Exogenous polyamines improve seed germination of borage under salt stress via involvement in antioxidant defenses. WALIA J. 31 57–63.

Shu S., Yuan L. Y., Guo S. R., Sun J., Liu C. J. (2012). Effects of exogenous spermidine on photosynthesis, xanthophyll cycle and endogenous polyamines in cucumber seedlings exposed to salinity. Afr. J. Biotechnol. 11 6064–6074. 10.5897/AJB11.1354 DOI

Shu S., Yuan Y., Chen J., Sun J., Zhang W., Tang Y., et al. (2015). The role of putrescine in the regulation of proteins and fatty acids of thylakoid membranes under salt stress. Sci. Rep. 5:14390. 10.1038/srep14390 PubMed DOI PMC

Silva A. T., Ligterink W., Hilhorst H. W. M. (2017). Metabolite profiling and associated gene expression reveal two metabolic shifts during the seed-to-seedling transition in Arabidopsis thaliana. Plant Mol. Biol. 95 481–496. 10.1007/s11103-017-0665-x PubMed DOI PMC

Talat A., Nawaz K., Hussian K., Bhatti K. H., Siddiqi E. H., Khalid A., et al. (2013). Foliar application of proline for salt tolerance of two wheat (Triticum aestivum L.) cultivars. World Appl. Sci. J. 22 547–554. 10.5829/idosi.wasj.2013.22.04.19570 DOI

Tanabata T., Shibaya T., Hori K., Ebana K., Yano M. (2012). SmartGrain: high-throughput phenotyping software for measuring seed shape through image analysis. Plant Physiol. 160 1871–1880. 10.1104/pp.112.205120 PubMed DOI PMC

Teh C. Y., Shaharuddin N. A., Ho C. L., Mahmood M. (2016). Exogenous proline significantly affects the plant growth and nitrogen assimilation enzymes activities in rice (Oryza sativa) under salt stress. Acta Physiol. Plant 38:151 10.1007/s11738-016-2163-1 DOI

Thiam M., Champion A., Diouf D., Mame Ourèye S. Y. (2013). NaCl effects on in vitro germination and growth of some senegalese cowpea (Vigna unguiculata (L.) Walp.) Cultivars. ISRN Biotechnol. 2013:382417. 10.5402/2013/382417 PubMed DOI PMC

Van Oosten M. J., Pepe O., De Pascale S., Silletti S., Maggio A. (2017). The role of biostimulants and bioeffectors as alleviators of abiotic stress in crop plants. Chem. Biol. Technol. Agric. 4:5 10.1186/s40538-017-0089-5 DOI

Yakhin O. I., Lubyanov A. A., Yakhin I. A., Brown P. H. (2016). Biostimulants in plant science: a global perspective. Front. Plant Sci. 7:2049. 10.3389/fpls.2016.02049 PubMed DOI PMC

Zeng D., Luo X., Tu R. (2012). Application of bioactive coatings based on chitosan for soybean seed protection. Int. J. Carbohydr. Chem. 2012 1–5. 10.1155/2012/104565 DOI

Sulfonation of IAA in Urtica eliminates its DR5 auxin activity

The loss-of-function of AtNATA2 enhances AtADC2-dependent putrescine biosynthesis and priming, improving growth and salinity tolerance in Arabidopsis

Presence and future of plant phenotyping approaches in biostimulant research and development

Addressing the contribution of small molecule-based biostimulants to the biofortification of maize in a water restriction scenario

Biostimulants as an Alternative to Improve the Wine Quality from Vitis vinifera (cv. Tempranillo) in La Rioja

Priming with Small Molecule-Based Biostimulants to Improve Abiotic Stress Tolerance in Arabidopsis thaliana

Seed Priming With Protein Hydrolysates Improves Arabidopsis Growth and Stress Tolerance to Abiotic Stresses

Integration of Phenomics and Metabolomics Datasets Reveals Different Mode of Action of Biostimulants Based on Protein Hydrolysates in Lactuca sativa L. and Solanum lycopersicum L. Under Salinity

Hormopriming to Mitigate Abiotic Stress Effects: A Case Study of N 9-Substituted Cytokinin Derivatives With a Fluorinated Carbohydrate Moiety

Understanding the Biostimulant Action of Vegetal-Derived Protein Hydrolysates by High-Throughput Plant Phenotyping and Metabolomics: A Case Study on Tomato