Tesi etd-03312020-180548
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Tipo di tesi
Dottorato
Autore
CARDINI, ALESSIO
URN
etd-03312020-180548
Titolo
Interplant communication mediated by arbuscular mycorrhizal fungal hyphal networks
Settore scientifico disciplinare
Istituto di Scienze della Vita
Corso di studi
Istituto di Scienze della Vita - AGROBIOSCIENCES
Commissione
relatore Prof.ssa ERCOLI, LAURA
Tutor Dott.ssa PELLEGRINO, ELISA
Membro Prof. FERROL, NURIA
Membro Prof. LANFRANCO, LUISA
Membro Prof. SEBASTIANI, LUCA
Tutor Dott.ssa PELLEGRINO, ELISA
Membro Prof. FERROL, NURIA
Membro Prof. LANFRANCO, LUISA
Membro Prof. SEBASTIANI, LUCA
Parole chiave
- Zinc
- micronutrient
- biofortification
- alfalfa (Medicago sativa)
- Nicotianamine
- Metal Tolerance Protein (MTP)
- Yellow Stripe-Like Protein (YSL)
- Zinc Induced facilitators (ZIF)
- Heavy Metal transporters (HMA)
- common mycorrhizal network
- Zn transfer
- Medicago truncatula
- Rhizophagus irregularis
- Cation Diffusion Facilitators (CDFs)
- Zinc-Iron-Regulated Transporter (ZRT-IRT)
- ZIP transporters
- digital image analysis
- plant-fungus phenotyping
- root length
- hyphal length
- arbuscular mycorrhizal fungi
- extraradical mycelium
Data inizio appello
03/06/2020;
Disponibilità
parziale
Riassunto analitico
Arbuscular mycorrhizal fungi (AMF) form one of the most widespread land symbiosis established with over 80% of terrestrial plant species. The extraradical mycelium of AMF is able to link together plants belonging to the same or different species, genera and families in a common mycorrhizal network (CMN) and is also able to mediate the transfer of nutrients and photosynthetic carbon within the soil and among the interconnected plants. The CMN is a complex structure, whose density varies depending on the identity of the AMF isolate and of the interconnected plant species. Some studies demonstrated both in laboratory and in field the transfer of macronutrients, such as P and N, from plant to plant through the CMN. However, the role played by the CMN in interplant transfer and redistribution of micronutrients, such as zinc (Zn), was not clarified, despite the proved positive roles of AMF in crop acquisition of micronutrients. Zinc is an essential micronutrient for the growth and health of plants and other organisms, such as bacteria and fungi, as well as for humans. Zinc deficiency reduces plant growth and production, because it is required for many physiological functions. Conversely, if accumulated in excess Zn can be toxic. As a result, plants have developed complex homeostatic mechanisms that ensure optimal cellular Zn concentration when grown in soil with limiting or toxic levels of Zn.
The aim of this PhD thesis was to provide new insight on foliar Zn uptake and distribution within plants and among plants by AMF in order to optimize the use of Zn fertilizers and the nutritional and detoxifying services of AMF. Additional aims were to develop new assays to study the gene expression of Zn transporters of plants and AMF, and to develop a new tool for plant and fungal phenotyping in order to efficiently measure the growth of roots and fungal hyphae. To this aims, three experiments were carried out:
(i) Study of the shoot-to-root redistribution of Zn after Zn foliar application in an in vivo experiment with Medicago sativa plants. Zn applications were 0, 0.01 (deficiency), 0.1 (optimum), 0.5, 1 (close to toxicity) and 10 mg Zn per plant (toxicity). The Zn allocation in shoot and root was assessed together with gene expression of twelve plant transporters involved in Zn plant redistribution. New qPCR assays were developed to study the gene expression of Zn transporters of M. sativa (MsZIP1-7, MsZIF1, MsMTP1, MsYSL1, MsHMA4 and MsNAS1) (Exp. 1).
(ii) Validation and benchmarking of a newly developed semi-automated digital image analysis tool, named HyLength, able to measure the length of roots and fungal hyphae of dense mycelia (e.g., AMF hyphae) grown in in vivo and in vitro systems. The tool was built using functions of the Image Processing Toolbox of MATLAB R2019b (Exp. 2).
(iii) Study of communication among plants mediated by the AM fungus Rhizophagus irregularis. In an in vitro system, the Zn transfer between two Medicago truncatula plants, linked by a CMN, was determined after Zn application on donor plant leaves. The Zn allocation in shoots and roots of donor and receiver plants was assessed, together with the expression of plant and fungal genes involved in Zn homeostasis (plant: MtZIP1, MtZIP2 and MtNAS1; fungus: RiZnT1 and RiZRT1). New qPCR assays were developed to study the gene expression of Zn transporters for R. irregularis (Exp. 3).
Results from exp. 1 demonstrated that Zn applied to leaves of M. sativa was translocated to roots. Zinc shoot and root concentration was increased following foliar Zn application higher or equal to 0.1 mg Zn per plant. Upregulation of MsZIP2, MsHMA4 and MsNAS1 in shoots and of MsZIP2 and MsHMA4 in roots were found under the highest Zn dose. By contrast, MsZIP3 was downregulated in shoots at Zn doses higher or equal to 0.1 mg Zn per plant. Significant increases of the expression levels of MsZIP2 suggest plant detoxification through the accumulation of Zn in the xylem parenchyma under excess of Zn after foliar application, whereas the decreases of the expression levels of MsZIP3 indicate a limitation of the Zn influx in the cell cytoplasm of shoots from vascular tissues. Moreover, the upregulation of MsHMA4 is involved in whole-plant Zn redistribution under toxicity conditions, while the corresponding upregulation of MsNAS1 suggests the chelation of the excess of Zn in the shoot tissues, enabling the redistribution to roots.
The HyLength tool was developed for measuring the lengths of hyphae and roots in in vivo and in vitro systems (exp. 2). HyLength was successfully validated against manual measures of roots and fungal hyphae. Compared to manual methods, HyLength underestimated M. sativa roots in the in vivo system and R. irregularis hyphae in the in vitro system by about 12 cm per meter and allowed to save about 1 hour for a single experimental unit. As regards hyphae of R. irregularis in the in vivo system, HyLength overestimated the length by about 21 cm per meter compared to manual measures, but time saving was up to 20.5 h per single experimental unit. Finally, with hyphae of Aspergillus oryzae the underestimation was about 8 cm per meter with a time saving of about 10 minutes for a single germinating spore. By benchmarking HyLength against the AnaMorf plugin of ImageJ/Fiji, we found that HyLength performed better for dense fungal hyphae also strongly reducing the measuring time. HyLength can allow measuring the length over a whole experimental unit, eliminating the error due to sub-area selection by the user and allowing processing a high number of samples.
After Zn application to leaves of M. truncatula donor plants, Zn was transferred to donor roots and then to the roots of receiver plants linked by the CMN of R. irregularis (exp. 3). The MtNAS1 gene was overexpressed in shoots and roots of donor plants as well as in the roots of the receiver plants, highlighting NA involvement in Zn mobilization within plants. The increased expression of the MtNAS1 also in receiver plant roots is the evidence of the movement of Zn to the root cells after Zn translocation due to the AM fungus. Indeed, the two fungal Zn transporter genes, RiZRT1 and RiZnT1, were overexpressed in the roots of donor and receiver plants, demonstrating the active role of AMF in Zn transfer between linked plants.
In conclusion, these researches gave new insights into the Zn homeostasis of M. sativa plants after foliar Zn application and on the transfer of Zn between plants interconnected by a fungal hyphal network. This knowledge could be agronomically relevant, since it helps to define effective biofortification strategies for forage crops, aiming to improve the quality of the feed, the health of livestock with potentially significant effects also on human health. Moreover, the findings on genes involved in Zn uptake, redistribution and sequestration of M. sativa could be used for engineering strategies, aiming to increase the efficiency of Zn application, the accumulation of Zn in edible plant parts as well as the tolerance of plants to Zn toxicity.
The aim of this PhD thesis was to provide new insight on foliar Zn uptake and distribution within plants and among plants by AMF in order to optimize the use of Zn fertilizers and the nutritional and detoxifying services of AMF. Additional aims were to develop new assays to study the gene expression of Zn transporters of plants and AMF, and to develop a new tool for plant and fungal phenotyping in order to efficiently measure the growth of roots and fungal hyphae. To this aims, three experiments were carried out:
(i) Study of the shoot-to-root redistribution of Zn after Zn foliar application in an in vivo experiment with Medicago sativa plants. Zn applications were 0, 0.01 (deficiency), 0.1 (optimum), 0.5, 1 (close to toxicity) and 10 mg Zn per plant (toxicity). The Zn allocation in shoot and root was assessed together with gene expression of twelve plant transporters involved in Zn plant redistribution. New qPCR assays were developed to study the gene expression of Zn transporters of M. sativa (MsZIP1-7, MsZIF1, MsMTP1, MsYSL1, MsHMA4 and MsNAS1) (Exp. 1).
(ii) Validation and benchmarking of a newly developed semi-automated digital image analysis tool, named HyLength, able to measure the length of roots and fungal hyphae of dense mycelia (e.g., AMF hyphae) grown in in vivo and in vitro systems. The tool was built using functions of the Image Processing Toolbox of MATLAB R2019b (Exp. 2).
(iii) Study of communication among plants mediated by the AM fungus Rhizophagus irregularis. In an in vitro system, the Zn transfer between two Medicago truncatula plants, linked by a CMN, was determined after Zn application on donor plant leaves. The Zn allocation in shoots and roots of donor and receiver plants was assessed, together with the expression of plant and fungal genes involved in Zn homeostasis (plant: MtZIP1, MtZIP2 and MtNAS1; fungus: RiZnT1 and RiZRT1). New qPCR assays were developed to study the gene expression of Zn transporters for R. irregularis (Exp. 3).
Results from exp. 1 demonstrated that Zn applied to leaves of M. sativa was translocated to roots. Zinc shoot and root concentration was increased following foliar Zn application higher or equal to 0.1 mg Zn per plant. Upregulation of MsZIP2, MsHMA4 and MsNAS1 in shoots and of MsZIP2 and MsHMA4 in roots were found under the highest Zn dose. By contrast, MsZIP3 was downregulated in shoots at Zn doses higher or equal to 0.1 mg Zn per plant. Significant increases of the expression levels of MsZIP2 suggest plant detoxification through the accumulation of Zn in the xylem parenchyma under excess of Zn after foliar application, whereas the decreases of the expression levels of MsZIP3 indicate a limitation of the Zn influx in the cell cytoplasm of shoots from vascular tissues. Moreover, the upregulation of MsHMA4 is involved in whole-plant Zn redistribution under toxicity conditions, while the corresponding upregulation of MsNAS1 suggests the chelation of the excess of Zn in the shoot tissues, enabling the redistribution to roots.
The HyLength tool was developed for measuring the lengths of hyphae and roots in in vivo and in vitro systems (exp. 2). HyLength was successfully validated against manual measures of roots and fungal hyphae. Compared to manual methods, HyLength underestimated M. sativa roots in the in vivo system and R. irregularis hyphae in the in vitro system by about 12 cm per meter and allowed to save about 1 hour for a single experimental unit. As regards hyphae of R. irregularis in the in vivo system, HyLength overestimated the length by about 21 cm per meter compared to manual measures, but time saving was up to 20.5 h per single experimental unit. Finally, with hyphae of Aspergillus oryzae the underestimation was about 8 cm per meter with a time saving of about 10 minutes for a single germinating spore. By benchmarking HyLength against the AnaMorf plugin of ImageJ/Fiji, we found that HyLength performed better for dense fungal hyphae also strongly reducing the measuring time. HyLength can allow measuring the length over a whole experimental unit, eliminating the error due to sub-area selection by the user and allowing processing a high number of samples.
After Zn application to leaves of M. truncatula donor plants, Zn was transferred to donor roots and then to the roots of receiver plants linked by the CMN of R. irregularis (exp. 3). The MtNAS1 gene was overexpressed in shoots and roots of donor plants as well as in the roots of the receiver plants, highlighting NA involvement in Zn mobilization within plants. The increased expression of the MtNAS1 also in receiver plant roots is the evidence of the movement of Zn to the root cells after Zn translocation due to the AM fungus. Indeed, the two fungal Zn transporter genes, RiZRT1 and RiZnT1, were overexpressed in the roots of donor and receiver plants, demonstrating the active role of AMF in Zn transfer between linked plants.
In conclusion, these researches gave new insights into the Zn homeostasis of M. sativa plants after foliar Zn application and on the transfer of Zn between plants interconnected by a fungal hyphal network. This knowledge could be agronomically relevant, since it helps to define effective biofortification strategies for forage crops, aiming to improve the quality of the feed, the health of livestock with potentially significant effects also on human health. Moreover, the findings on genes involved in Zn uptake, redistribution and sequestration of M. sativa could be used for engineering strategies, aiming to increase the efficiency of Zn application, the accumulation of Zn in edible plant parts as well as the tolerance of plants to Zn toxicity.
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