Periodic Reporting for period 1 - PlantIMS (Fast and Sensitive Detection of Plant Hormones by Ion Mobility Spectrometry)
Periodo di rendicontazione: 2021-07-01 al 2023-06-30
Problem/issue being addressed:
The plant hormones (PHs), are signal molecules produced within plants that occur at extremely low concentration. PHs influence the plant growth, seed germination, fruit maturation and fruit ripening, they have significant roles in the production of food crops. They are responsible for controlling the physiological processes including the embryogenesis, regulation of the organ size, pathogen defence, and reproductive developments. Therefore, an in-depth understanding of PHs and their deliberate incorporation into the food crop production application can lead to remarkable results helping to tackle climate changes and food crops health.
According to the Food and Agriculture Organization (FAO) achieving the food security and improved nutrition for the growing world population while promoting sustainable agriculture is one of the sustainable development goals. The research in the field of PHs can contribute to achieve these goals in the agriculture, such as inhibition of invasive parasitic plants in sub-Saharan
region, or to increase the drought tolerance of wheat. This project can contribute to the sustainable food crop production and relieving the concerns about food security. Starting with development of new fast and cost-effective quantitative PHs detection method.
The primary goal of the project was exploration of IMS technique for fast and sensitive detection of PHs and to develop sampling methods and methodology for online measurement of PHs released from plants. The goals of the project have been achieved. The methods based in IMS, MCC and SPME sampling methods have been shown as fast and sensitive method of detection of plant hormones.
The plant hormones (PHs), are signal molecules produced within plants that occur at extremely low concentration. PHs influence the plant growth, seed germination, fruit maturation and fruit ripening, they have significant roles in the production of food crops. They are responsible for controlling the physiological processes including the embryogenesis, regulation of the organ size, pathogen defence, and reproductive developments. Therefore, an in-depth understanding of PHs and their deliberate incorporation into the food crop production application can lead to remarkable results helping to tackle climate changes and food crops health.
According to the Food and Agriculture Organization (FAO) achieving the food security and improved nutrition for the growing world population while promoting sustainable agriculture is one of the sustainable development goals. The research in the field of PHs can contribute to achieve these goals in the agriculture, such as inhibition of invasive parasitic plants in sub-Saharan
region, or to increase the drought tolerance of wheat. This project can contribute to the sustainable food crop production and relieving the concerns about food security. Starting with development of new fast and cost-effective quantitative PHs detection method.
The primary goal of the project was exploration of IMS technique for fast and sensitive detection of PHs and to develop sampling methods and methodology for online measurement of PHs released from plants. The goals of the project have been achieved. The methods based in IMS, MCC and SPME sampling methods have been shown as fast and sensitive method of detection of plant hormones.
Explanation of the work carried out
1. Detection of auxin plant hormones by ion mobility-mass spectrometry:
Auxins are an important class of plant hormones that control plant growth, root initiation, and fruit growth and development. Measurements were carried out by IMS and time of flight mass spectrometry (TOFMS) for three auxins including indole-3-acetic acid (IAA), indole-3-propionic acid (IPA), and indole-3-butyric acid (IBA) in both positive and negative polarities of the corona discharge ion source. Dopant gases were used to modify the ionization mechanism.The results of this project were published in the journal Analytical and bioanalytical Chemistry (https://link.springer.com/article/10.1007/s00216-022-04198-x(si apre in una nuova finestra)). One of the important results of this study was that the dopant and gas modifier dramatically improve the analysis of a mixture of auxin plant hormones. (Figure 1).
2. Detection of Methyl Salicylate plant hormone in Tomato Leaves:
Methyl salicylate (MeSA) is a plant-signalling molecule that plays an essential role in the regulation of the plant responses to biotic and abiotic pathogens. The solid phase microextraction (SPME) sampling and multi-capillary column (MCC) was coupled to ion mobility spectrometry (IMS) to measure MeSA in tomato leaves (Figure 2). The SPME-MCC-IMS method provides two-dimensional (2D) separation by both MCC and IMS, basis on the drift time and retention time. The results of this project were published in the Journal of Agricultural and Food Chemistry (https://pubs.acs.org/doi/abs/10.1021/acs.jafc.2c05570(si apre in una nuova finestra)). The results showed that the SPME-MCC-IMS technique can be applied not only for qualitative and quantitative analysis of MeSA but also for other volatile compounds released from plants.
3. Introduction of formic acid as a dopant for analysis of plant hormones:
Cytokines and auxins are two important classes of plant hormones, with completely different chemical structures. In this study, formic acid (FA) was introduced as a potent dopant for APCI ionization in the negative polarity to enhance sensitivity and selectivity of IMS toward detection of auxins and cytokinins plant hormones. The schematic representation of the experimental set-up for the measurements with FA dopant is shown in Figure 3. Qualitative and quantitative analysis of the cytokinin and auxin plant hormones showed that FA dopant leads to the improvement of detection sensitivity, suppression of fragmentation, and changes in the ion mobilities of the analytes. The results of this project were accepted for publication in the Journal of American Society for Mass Spectrometry (https://doi.org/10.1021/jasms.3c00225(si apre in una nuova finestra)).
4. Measurement of cytokinins in coconut juice by LC-IM-QTOF:
In this study, three cytokinins including zeatin, kinetin, 6-Benzylaminopurine (BAP) were measured in coconut juice in collaboration with BOKU university in Vienna. An commercial Agilent 6560 LC-IM-QTOF mass spectrometer with a dual jetstream electrospray ion source (ESI) was used for ionisation of the compounds. A solid phase extraction (SPE) column (HyperSep C18) was use for extraction cytocinones from coconut water. Figure 4 shows the LC-chromatogram of a mixture of the three cytokinins at different concentration and the calibration curves for the cytokinins obtained from the chromatograms. The manuscript of this work is in preparation.
5. In-site measurement of volatile plant hormones in tomato and blubbery:
Plants emit some volatiles compounds and plant hormones when they are exposed to different pathogens and mechanical damages. The emitted plant hormones from blubbery and tomato were studied. A device with different components for collecting the chemicals was designed and constructed (Figure 5a to f). The sampling time using a Tenax sampler was set to 20 min. To desorb the adsorbed compounds desorption unit (range of 25-300 °C) was designed and constructed (Figure 6). Samples were analysed by MMC-IMS technique. The PHs relese from plants was studied and analysed: (i) after inoculation by the virus, (ii) after mechanical punching of the leaves, and (iii) after exposing the plants to insects. In the last case, the emitted compounds are called herbivore-induced plant volatiles (HIPVs). The result of this project are in preparation to be submitted to the Journal of Chromatography A.
1. Detection of auxin plant hormones by ion mobility-mass spectrometry:
Auxins are an important class of plant hormones that control plant growth, root initiation, and fruit growth and development. Measurements were carried out by IMS and time of flight mass spectrometry (TOFMS) for three auxins including indole-3-acetic acid (IAA), indole-3-propionic acid (IPA), and indole-3-butyric acid (IBA) in both positive and negative polarities of the corona discharge ion source. Dopant gases were used to modify the ionization mechanism.The results of this project were published in the journal Analytical and bioanalytical Chemistry (https://link.springer.com/article/10.1007/s00216-022-04198-x(si apre in una nuova finestra)). One of the important results of this study was that the dopant and gas modifier dramatically improve the analysis of a mixture of auxin plant hormones. (Figure 1).
2. Detection of Methyl Salicylate plant hormone in Tomato Leaves:
Methyl salicylate (MeSA) is a plant-signalling molecule that plays an essential role in the regulation of the plant responses to biotic and abiotic pathogens. The solid phase microextraction (SPME) sampling and multi-capillary column (MCC) was coupled to ion mobility spectrometry (IMS) to measure MeSA in tomato leaves (Figure 2). The SPME-MCC-IMS method provides two-dimensional (2D) separation by both MCC and IMS, basis on the drift time and retention time. The results of this project were published in the Journal of Agricultural and Food Chemistry (https://pubs.acs.org/doi/abs/10.1021/acs.jafc.2c05570(si apre in una nuova finestra)). The results showed that the SPME-MCC-IMS technique can be applied not only for qualitative and quantitative analysis of MeSA but also for other volatile compounds released from plants.
3. Introduction of formic acid as a dopant for analysis of plant hormones:
Cytokines and auxins are two important classes of plant hormones, with completely different chemical structures. In this study, formic acid (FA) was introduced as a potent dopant for APCI ionization in the negative polarity to enhance sensitivity and selectivity of IMS toward detection of auxins and cytokinins plant hormones. The schematic representation of the experimental set-up for the measurements with FA dopant is shown in Figure 3. Qualitative and quantitative analysis of the cytokinin and auxin plant hormones showed that FA dopant leads to the improvement of detection sensitivity, suppression of fragmentation, and changes in the ion mobilities of the analytes. The results of this project were accepted for publication in the Journal of American Society for Mass Spectrometry (https://doi.org/10.1021/jasms.3c00225(si apre in una nuova finestra)).
4. Measurement of cytokinins in coconut juice by LC-IM-QTOF:
In this study, three cytokinins including zeatin, kinetin, 6-Benzylaminopurine (BAP) were measured in coconut juice in collaboration with BOKU university in Vienna. An commercial Agilent 6560 LC-IM-QTOF mass spectrometer with a dual jetstream electrospray ion source (ESI) was used for ionisation of the compounds. A solid phase extraction (SPE) column (HyperSep C18) was use for extraction cytocinones from coconut water. Figure 4 shows the LC-chromatogram of a mixture of the three cytokinins at different concentration and the calibration curves for the cytokinins obtained from the chromatograms. The manuscript of this work is in preparation.
5. In-site measurement of volatile plant hormones in tomato and blubbery:
Plants emit some volatiles compounds and plant hormones when they are exposed to different pathogens and mechanical damages. The emitted plant hormones from blubbery and tomato were studied. A device with different components for collecting the chemicals was designed and constructed (Figure 5a to f). The sampling time using a Tenax sampler was set to 20 min. To desorb the adsorbed compounds desorption unit (range of 25-300 °C) was designed and constructed (Figure 6). Samples were analysed by MMC-IMS technique. The PHs relese from plants was studied and analysed: (i) after inoculation by the virus, (ii) after mechanical punching of the leaves, and (iii) after exposing the plants to insects. In the last case, the emitted compounds are called herbivore-induced plant volatiles (HIPVs). The result of this project are in preparation to be submitted to the Journal of Chromatography A.
Main innovation outputs (if applicable)
- Development of a fast and cost-efficient method for in-site analysis of VOCs emitted from plants by coupling Tenax-MCC-IMS (total analysis time 30 min).
- Development a fast method for analysis of MeSA plant hormone in plant leaves by coupling SPME-MCC-IMS (total analysis time 5 min).
- Introduction of formic acid as a new dopant not only for analysis of plant hormones but also as general dopant for the negative polarity of the chemical ionization sources of ion mobility and mass spectrometry to achieve higher sensitivity and reduced fragmentation.
- Development a new method for targeted and untargeted analysis using protomer-dependant fragmentation.
Contribution to the state of the art
- Introduction of the MCC-IMS technology to the field of plant analysis and phytochemistry.
- The SPME-MCC-IMS method was developed for detection of MeSA in tomato.
- An LC-IM-QTOF was developed for measurement of cytokinins in coconut water based on protomer-dependant fragmentation. This method can be used in the field of untargeted analysis.
- An online method based on Tenax adsorbent was developed for in-site detection of the plant hormones emitted from tomato and blueberry plant.
- Formic acid was introduced as a new dopant for using in the negative mode of IMS and MS.
- Development of a fast and cost-efficient method for in-site analysis of VOCs emitted from plants by coupling Tenax-MCC-IMS (total analysis time 30 min).
- Development a fast method for analysis of MeSA plant hormone in plant leaves by coupling SPME-MCC-IMS (total analysis time 5 min).
- Introduction of formic acid as a new dopant not only for analysis of plant hormones but also as general dopant for the negative polarity of the chemical ionization sources of ion mobility and mass spectrometry to achieve higher sensitivity and reduced fragmentation.
- Development a new method for targeted and untargeted analysis using protomer-dependant fragmentation.
Contribution to the state of the art
- Introduction of the MCC-IMS technology to the field of plant analysis and phytochemistry.
- The SPME-MCC-IMS method was developed for detection of MeSA in tomato.
- An LC-IM-QTOF was developed for measurement of cytokinins in coconut water based on protomer-dependant fragmentation. This method can be used in the field of untargeted analysis.
- An online method based on Tenax adsorbent was developed for in-site detection of the plant hormones emitted from tomato and blueberry plant.
- Formic acid was introduced as a new dopant for using in the negative mode of IMS and MS.