Abstract
Sustainable and environmentally friendly agriculture represents the main strategy to meet rising food needs and to contribute to a stable planet. To achieve these goals, one of the most promising solutions is the adoption of plant biostimulants. Biostimulants are natural compounds which, at low concentrations, are able to enhance plant nutrient uptake, nutrient efficiency, tolerance to abiotic/biotic stresses, and crop yield. Humic substances (HS) fall within the biostimulants definition, as they have positive effects on the uptake of macro and micronutrients that considerably improve the metabolism, the growth and yields of relevant agricultural crops. With this background, the aims of this Ph.D. project were to i) evaluate metabolic and biochemical status of plants treated with HS to elucidate the action mechanisms of plant biostimulants. This might permit the development of a second generation of biostimulants where synergies and complementary mechanism can be functionally designed. ii) investigate potential biostimulant effects of a fungicide compound. In manuscript I Arabidopsis thaliana plants were treated with HS for eight hours. Root proteins were studied by MS spectrometry coupled with iTRAQ (Isobaric Tag for Relative and Absolute Quantification) technique. 902 protein spots were identified for HS treated roots, where 291 proteins were differentially expressed. Bioinformatic tools such as DAVID, KEGG, IIS and Cytoscape were used to interpret the biological function, pathway analysis and visualization of the network amongst the identified proteins. From this analysis, it was possible to evaluate that most of the differentially expressed proteins were functionally classified into response to inorganic substances, redox homeostasis, energy metabolism, protein synthesis, cell trafficking and division. With the present study an overview of the metabolic pathways most modified by HS biological activity is provided. Moreover, from the analysis of interactomes and DAVID clusters it was possible to observe previously undiscovered HS effects, i.e. on the Ubiquitin and RACK1A interactome subnetworks. In manuscript II Arabidopsis thaliana plants, grown in hydroponic conditions, were treated for 8 h with indole-3-acetic acid (IAA), HS from International Humic Substances Society (IHSS) and HS from earthworm faeces (EF), respectively. Humic substances structural characteristics were analysed by 1H NMR an FT-IR spectroscopies. Root and leaf free amino acids, sugar alcohols and carbohydrates contents, and leaf amino acids from protein hydrolysis were identified and quantify by gas chromatography-mass spectrometry (GC/MS), and liquid chromatography-mass spectrometry (LC/MS) techniques. Canonical discriminant analysis (CDA) was used to evaluate the influence of the treatments on the studied parameters. EF treatment had the highest influence on metabolites profiles compared to the control, IAA and IHSS. CDA analysis highlighted a clear distinction between EF and IHSS plant physiological responses, depending on the different chemical and structural properties of the HS. IAA-treated plants resulted not significant different from the control. A better understanding of the specific effects of different HS, also related to their chemical characteristics, might serve as a basis for the identification of marker compounds for HS bioactivity. In manuscript III the biostimulant activity of HS extracted from four leonardites is analysed on maize seedlings. After 48 h of treatment with five concentrations (0, 0.1, 0.5, 1, and 10 mg C L−1) of HS, root growth and morphology, glutamine synthetase (GS) activity, glutamate synthase (GOGAT) activity, total protein content, soluble sugars content, phenylalanine ammonia-lyase (PAL) activity, soluble phenols, and free phenolic acids were analyzed. HS from different leonardites had similar spectroscopic pattern, with small differences. HS_USA best enhanced total root growth, root surface area, and proliferation of secondary roots. Plant nutrient use efficiency was enhanced by HS_4, HS_USA and HS_B, with increment of GS and GOGAT enzymes activity and total protein production. HS stimulated also PAL enzyme activity, followed by a higher production of total soluble phenols. This study found that, although the activity of the HS depended on the origin of the leonardite, these compounds can be attributed to the biostimulant products, eliciting plant growth, nitrogen metabolism, and accumulation of phenolic substances. In the manuscript IV it was investigated the potential biostimulant side-effects of sedaxane. Physiological changes in disease-free maize seedlings at increasing application doses (25, 75 and 150 μg a.i. seed-1) under controlled sterilised conditions were analysed. Sedaxane had significant auxin-like and gibberellin-like effects, with maximum benefits attained at the intermediate dose. Root length (+60% vs. untreated controls), area (+45%) and forks (+51%) were significantly increased. Sedaxane enhanced leaf and root glutamine synthetase (GS) activity resulting in greater protein accumulation. Sedaxane also improved leaf phenylalanine ammonia-lyase (PAL) activity, which may be responsible for the increase in shoot antioxidant activity (phenolic acids). It is concluded that, in addition to its protective effect, sedaxane can facilitate root establishment and intensify nitrogen and phenylpropanoid metabolism in young maize plants, and may be beneficial in overcoming biotic and abiotic stresses in early growth stages. As a general conclusion, even if HS were extracted from different sources were applied on different plant species, they displayed similar biological activities. Proteomics and metabolomics studies confirmed that “–omics” techniques are essential tools to have a ‘panoramic’ view on metabolic changes happening inside an organism after a positive or negative external perturbation.
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Claims 18
Claim · model-written summary
Humic substances changed expression of 291 root proteins in Arabidopsis.
- Taxon
- Arabidopsis thaliana
Arabidopsis thalianae1 - Organ / stage
- rootse0
- Treatment
- HSe1
- Outcome
- proteinse0
- Direction
- differentially expressede0
- Value
- 291 proteinse0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“902 protein spots were identified for HS treated roots, where 291 proteins were differentially expressed”
e1“Arabidopsis thaliana plants were treated with HS for eight hours”
Claim · model-written summary
Humic substances affected Arabidopsis ubiquitin and RACK1A interaction subnetworks.
- Taxon
- Arabidopsis thalianaArabidopsis thalianae1
- Treatment
- HSe1
- Outcome
- Ubiquitin and RACK1A interactome subnetworkse0
Not reportedComparatorDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“previously undiscovered HS effects, i.e. on the Ubiquitin and RACK1A interactome subnetworks”
e1“Arabidopsis thaliana plants were treated with HS for eight hours”
Claim · model-written summary
Earthworm-faeces humic substances had the greatest influence on Arabidopsis metabolite profiles.
- Taxon
- Arabidopsis thalianaArabidopsis thalianae1
- Treatment
- EF treatmente0
- Outcome
- metabolites profilese0
- Direction
- highest influencee0
- Comparator
- the control, IAA and IHSSe0
- Setting
- hydroponic conditionse1
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“EF treatment had the highest influence on metabolites profiles compared to the control, IAA and IHSS”
e1“Arabidopsis thaliana plants, grown in hydroponic conditions”
Claim · model-written summary
EF and IHSS treatments produced distinct Arabidopsis physiological responses.
- Taxon
- Arabidopsis thalianaArabidopsis thalianae1
- Treatment
- EFe0
- Outcome
- plant physiological responsese0
- Direction
- clear distinctione0
- Comparator
- IHSSe0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“a clear distinction between EF and IHSS plant physiological responses”
e1“Arabidopsis thaliana plants, grown in hydroponic conditions”
Claim · model-written summary
IAA-treated Arabidopsis did not differ significantly from controls in the studied metabolite response.
- Taxon
- Arabidopsis thalianaArabidopsis thalianae1
- Treatment
- IAAe0
- Outcome
- plantse0
- Direction
- not significant differente0
- Comparator
- the controle0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“IAA-treated plants resulted not significant different from the control”
e1“Arabidopsis thaliana plants, grown in hydroponic conditions”
Claim · model-written summary
US-sourced humic substances enhanced maize total root growth, root surface area and secondary-root proliferation.
- Treatment
- HS_USAe0
- Outcome
- total root growth, root surface area, and proliferation of secondary rootse0
- Direction
- best enhancede0
Zea maysNot reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“HS_USA best enhanced total root growth, root surface area, and proliferation of secondary roots”
e1“HS extracted from four leonardites is analysed on maize seedlings”
Claim · model-written summary
HS_4 enhanced maize nutrient use efficiency with increased GS and GOGAT activity and protein production.
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Plant nutrient use efficiency was enhanced by HS_4, HS_USA and HS_B, with increment of GS and GOGAT enzymes activity and total protein production”
e1“HS extracted from four leonardites is analysed on maize seedlings”
Claim · model-written summary
HS_USA enhanced maize nutrient use efficiency with increased GS and GOGAT activity and protein production.
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Plant nutrient use efficiency was enhanced by HS_4, HS_USA and HS_B, with increment of GS and GOGAT enzymes activity and total protein production”
e1“HS extracted from four leonardites is analysed on maize seedlings”
Claim · model-written summary
HS_B enhanced maize nutrient use efficiency with increased GS and GOGAT activity and protein production.
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Plant nutrient use efficiency was enhanced by HS_4, HS_USA and HS_B, with increment of GS and GOGAT enzymes activity and total protein production”
e1“HS extracted from four leonardites is analysed on maize seedlings”
Claim · model-written summary
Humic substances stimulated maize PAL activity.
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“HS stimulated also PAL enzyme activity”
e1“HS extracted from four leonardites is analysed on maize seedlings”
Claim · model-written summary
Humic substances increased maize total soluble phenol production.
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“followed by a higher production of total soluble phenols”
e1“HS stimulated also PAL enzyme activity”
e2“HS extracted from four leonardites is analysed on maize seedlings”
Claim · model-written summary
Sedaxane had auxin-like and gibberellin-like effects on maize, with the greatest benefit at the intermediate dose.
- Treatment
- Sedaxanee0
- Outcome
- auxin-like and gibberellin-like effectse0
- Direction
- maximum benefitse0
- Qualifier
- intermediate dosee0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Sedaxane had significant auxin-like and gibberellin-like effects, with maximum benefits attained at the intermediate dose”
e1“disease-free maize seedlings at increasing application doses”
Claim · model-written summary
Sedaxane increased maize root length by 60% versus untreated controls.
- Treatment
- Sedaxanee2
- Outcome
- Root lengthe0
- Direction
- increasede0
- Value
- +60%e0
- Comparator
- untreated controlse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Root length (+60% vs. untreated controls), area (+45%) and forks (+51%) were significantly increased”
e1“disease-free maize seedlings at increasing application doses”
e2“Sedaxane had significant auxin-like and gibberellin-like effects”
Claim · model-written summary
Sedaxane increased maize root area by 45%.
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Root length (+60% vs. untreated controls), area (+45%) and forks (+51%) were significantly increased”
e1“disease-free maize seedlings at increasing application doses”
e2“Sedaxane had significant auxin-like and gibberellin-like effects”
Claim · model-written summary
Sedaxane increased maize root forks by 51%.
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Root length (+60% vs. untreated controls), area (+45%) and forks (+51%) were significantly increased”
e1“disease-free maize seedlings at increasing application doses”
e2“Sedaxane had significant auxin-like and gibberellin-like effects”
Claim · model-written summary
Sedaxane enhanced maize leaf and root glutamine synthetase activity and protein accumulation.
- Treatment
- Sedaxanee0
- Gene
- glutamine synthetase (GS)e0
- Outcome
- leaf and root glutamine synthetase (GS) activitye0
- Direction
- enhancede0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Sedaxane enhanced leaf and root glutamine synthetase (GS) activity resulting in greater protein accumulation”
e1“disease-free maize seedlings at increasing application doses”
Claim · model-written summary
Sedaxane improved maize leaf PAL activity.
- Treatment
- Sedaxanee0
- Gene
- phenylalanine ammonia-lyase (PAL)e0
- Outcome
- leaf phenylalanine ammonia-lyase (PAL) activitye0
- Direction
- improvede0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Sedaxane also improved leaf phenylalanine ammonia-lyase (PAL) activity”
e1“disease-free maize seedlings at increasing application doses”
Claim · model-written summary
Sedaxane increased maize shoot antioxidant activity, attributed tentatively to PAL activity.
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“which may be responsible for the increase in shoot antioxidant activity (phenolic acids)”
e1“Sedaxane also improved leaf phenylalanine ammonia-lyase (PAL) activity”
e2“disease-free maize seedlings at increasing application doses”
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See all 10 papers →Claims are grouped by organism, treatment and what was measured or targeted. A paper counts as positive when any of its claims in the group is positive.