Unraveling Regulated Cell Death in Plants Challanges and Perspectives using Mass Spectrometry Imaging

Abstract

N ̊ar klimaendringene akselererer og den globale temperaturen øker, utsettes planter for sterkere miljøbelastninger. Planter reagerer p ̊a stress ved ̊a aktivere ulike typer fysiologiske en- dringer i rotmorfologi og molekylære responser, og en slik respons er programmert celledød. Pro- grammert celledød er en kontrollert respons hvor skadede celler elimineres og fjernes. Dette er en viktig mekanisme, som gjør planter i stand til ̊a takle miljøbelastninger. Det ̊a forst ̊a mekanis- mene som regulerer programmert celledød er derfor et viktig felt i plantebiologien. En sentral del av dette er ̊a forst ̊a de molekylære endringene som oppst ̊ar kort tid etter eksponering for stress. Dette har til n ̊a vært vanskelig grunnet mangelen p ̊a metoder som tillater den spatiotemporale oppløsningen, men ny teknologi som MALDI-MSI gjør det n ̊a mulig ̊a foreta denne typen studier. MALDI-MSI er en molekylær avbildningsteknologi, som muliggjør identifisering og lokalisering av spesifikke molekyler i en prøve. Denne teknologien er tatt i bruk i det medisinske feltet, men er lite utprøvd p ̊a plantematerialet. M ̊alet med denne oppgaven har derfor vært ̊a undersøke de molekylære spatiotemporale endringene forbundet med programmert celledød ved ̊a eksponere arabidopsis thaliana-røtter for lav pH, samt ̊a fremme integreringen av massespektrometrisk avb- ildning for plantebaserte studier. Den første delen av oppgaven gikk ut p ̊a ̊a undersøke effekten av ulike buffere og pH p ̊a celledød. Spirer av arabidopsis ble eksponert for ulike buffere (NaAc, MES og Pipes/CaCl2) med pH 5.8 (normal) og 4.6 (lav) og eventuell celledød p ̊avist ved hjelp av Evans Blue og SYTOX green. Eksponering for NaAc-buffere førte til celledød b ̊ade ved pH 5.8 og 4.6. Ved pH 5.8 dannet plantene tilfeldige røtter, mens ved pH 4.6 ble de bleke. Dette indikerer at NaAc-buffer induserer celledød selv ved tilsynelatende normale pH-verdier s ̊a vel som ved lav pH. Dette var ogs ̊a tilfellet for MES-buffer, en buffer mye brukt i plantedyrking. Det kan dermed tyde p ̊a at MES-buffer ikke er en egnet buffer for planter, og ytterligere studier trengs for ̊a kartlegge den fysiologiske effekten. Eksponering for Pipes/CaCl2 derimot, gav ingen detekterbar celledød. M ̊alet med den andre delen av dette prosjektet var ̊a etablere MALDI-MSI for plantestudier, inkludert preparering av prøver. Dette var et krevende arbeid, som leder til konklusjonen om at en omfattende metodeutvikling og testing er nødvendig for ̊a oppn ̊a reproduserbare resultater. Samlet sett understreker resultatene fra denne oppgaven behovet for studier som undersøker buffer- induserte effekter og forskjeller mellom dem, samt fremhever noen av utfordringene og potensielle løsninger for ytterligere ̊a inkorporere MALDI-MSI for plantebaserte studier.

As climate change accelerates and global temperatures increase, plants are subjec- ted to stronger environmental stressors. Plants respond to stress by activating different types of physiological changes in root morphology and molecular responses, one such response is regulated cell death. Regulated cell death is a controlled response which eliminates and removes damaged cells. It is an important preservation mechanism as without it plants would be unable to recover and overcome exposure to environmental stressors. Therefore, research investigating the mech- anisms by which regulated cell death occurs in response to altered environmental conditions has become an important topic in the field of plant biology. One key part of this is understanding the molecular changes that occur shortly after stress exposure. This was previously difficult due to the lack of methodologies capable of reaching the necessary spatiotemporal resolution. With recent technological advances, equipment with such capabilities has been developed and has seen successful use in the medical field. One such methodology is MALDI-MSI, which allows the user to observe molecules present within a sample as well generating images illustrating the localization of specific masses within the sample. However, it is necessary to develop protocols and overcome several challenges to successfully incorporate this technology for use in plant research. Therefore, the purpose of this thesis was to investigate the molecular spatiotemporal changes associated with regulated cell death upon exposure to low pH in arabidopsis thaliana roots, as well as furthering the integration of mass spectrometry imaging for plant based studies. In an attempt to induce a localised cell death response, arabidopsis seedlings were exposed to various buffers (NaAc, MES and Pipes/CaCl2) at two pH conditions (Normal: 5.8 and Low: 4.6). Exposure to NaAc buffers led to Evans Blue and SYTOX green cell death patterns indicative of primary root death. This was further confirmed with the observation that seedlings also developed adventitious roots at pH: 5.8. Seedlings at pH: 4.6 exhibited bleaching and complete cell death, indicating NaAc buffer had induced toxicity effects at seemingly “normal” pH values as well as in low pH conditions. Interestingly, in MES buffers which are extensively used in plant cultivation, cell death was observed at both normal and low pH values as well as various tested concentrations and time points. These results suggest MES buffer may not be a suitable control for experiments and further research investigating its effects on plant physiology should be conducted. Contrastingly, Pipes/CaCl2 treated seedlings exhibited no discernable cell death patterns and were seemingly unaffected by buffer exposure. The second part of this project was focused on furthering the integration of mass spectrometry imaging technology for plant studies. For this, sample preparation and cryosectioning steps were conducted before mass spectrometry analyses. Difficulties encountered in the aforementioned steps led to the conclusion that extensive method development and testing is required in order to obtain reproducible results. Taken together, the results generated from this thesis emphasise the need for studies investigating buffer induced effects and differences between them, as well as highlighting some of the challenges and potential solutions to further incorporate MALDI-MSI for plant based studies.