Woody biochar, a promising mitigation strategy on camium stress on Arabidopsis thaliana growth

Abstract

Worldwide soils are polluted with cadmium (Cd) and this metal affects human health. Dietary uptake of Cd results in several human health problems such as renal and bone damage. Although non-essential for plants, Cd is taken up by the roots due to its chemical similarity with other essential elements, including calcium (Ca) and magnesium (Mg). Once taken up, Cd will elevate the level of reactive oxygen species (ROS). These molecules have a distinct signalling function and are able to trigger the accompanying networks when the balance between the pro and antioxidants in the plant is disrupted. However, high ROS levels can disrupt the balance between pro- and antioxidants, thereby inflicting damage to the plant. Several macromolecules can be affected, including proteins, fatty acids and DNA. Upon perceiving DNA damage, the DNA damage response (DDR) will be activated. This Cd-induced DDR is regulated by the transcription factor SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1). Activation of SOG1 is initiated upon phosphorylation by the cell cycle checkpoint kinases ATAXIA TELANGIECTASIA MUTATED (ATM) and RAD3-RELATED (ATR). Both ATM and ATR are activated upon perceiving DNA damage and are able to induce the expression of DNA repair genes via a downstream pathway thereby repairing these defects. ATM is able to repair double strand breaks (DSB), whereas ATR repairs single strand breaks (SSB) and replication fork defects. Furthermore, SOG1 also regulates the activation of several cell cycle genes, thereby affecting the cell cycle regulation. Cadmium is also known to affect cell cycle regulation, as it inhibits both cell division and endoreplication. The transcription factor SOG1 regulates the expression of certain cyclin-dependent kinases (CDKs) and they also play a role in the regulation of cell cycle progression by binding to a cyclin (CYC). The CDK-CYC complexes become active and thereby take part in affecting the regulation of the cell cycle. The function of these complexes can be inhibited due to the action of cyclin dependent inhibitors (CKI). Two CKI families can be found in plants, the SIAMESE-RELATED (SMRs) and the KIP related proteins (KRP). During a standard cell cycle, DNA is replicated and the cell division process will divide this DNA over two identical daughter cells. An alternative can be found in endoreplication, where endopolyploidy of the cell occurs due to the skipping of mitosis. In Arabidopsis thaliana (A. thaliana), ploidy levels of up to 64C (with C the haploid DNA content of the cell) have been reported. Endoreplication plays a role in plant cell growth, although research also observed that this process is involved in defence mechanisms of the plant. Upon perceiving stress, such as Cd induced damage, this creates a growth/defence trade-off. Furthermore, this trade-off is variable depending on for example plant age, tissue or cultivation system. However, thus far no analyses or measurements existed that enabled to compute this trade-off. Therefore, we calculated several flow cytometric indices: (1) nuclei concentration, (2) endoreplication index, (3) growth index and (4) defence index in hydroponically grown wild-type (WT) A. thaliana rosettes and individual leaves. Plants were exposed to 0, 1 or 5 µM CdSO4 to investigate which individual leaf best reflects the Cd-induced effects on the regulation of the DDR and growth arrest at cellular level in the whole rosette. This will help to correctly interpret the rosette data in the framework of plant development in further research and give further insight in the Cd-induced growth/defence trade-off (Chapter 3). To prevent the occurrence of Cd-induced human health problems and thus Cd uptake by crops, a solution needs to be found that enables to reduce the phytotoxicity of Cd. The need for a fast and controlled screening method, which enables research on several compounds, is imminent. We present the SAFETY96 cultivation method as a promising screening method. This method allows the cultivation of seedlings in 96-well plates filled with liquid growth medium. Several treatmentsor compounds can be added to the medium in order to screen these components and their interaction. The use of 96-well plates is space- and time efficient and enables the simultaneously testing of several conditions. To demonstrate this method, WT A. thaliana seeds were exposed to 0, 10 and 25 µM Cd and category A and B wood waste (AB), Pinus sylvestris tree bark (TB) or medium-density fiberboard (MDF) woody biochar, immediately after sowing. This enabled the study of the effect of the carbon-rich woody biochars on the Cd induced growth reduction. Preliminary results showed that biochar is a promising compound for reducing the Cd phytotoxicity. The optimal biochar type and concentration can be selected for the experiments using this screening method. The potential of woody biochar as a Cd-reducing agent was further studied in seedlings and substrate in order to compare the effects and differences of the woody biochars in detail (Chapter 4). It was observed that each woody biochar can mitigate the Cd-induced decrease of growth parameters but to varying degrees. Furthermore, woody biochars reduced the level of Cd-induced oxidative stress. This positive effect of woody biochar was further studied via flow cytometric parameters, which confirmed the positive effect on plant growth. This concept was repeated and thereby extrapolated in another, more field-relevant, cultivation system: pot experiments with sand-perlite substrate (1:1). This cultivation system provided a more realistic growth matrix while still being able to strictly control the experiments. Phenotypical analyses showed that woody biochar is able to reduce Cd phytotoxicity in the plant. This study thereby provided further insight into the capability of each woody biochar for amelioration of the Cd phytotoxicity in A. thaliana. The ¼ MS liquid growth medium and substrate were therefore used to grow WT A. thaliana plants exposed to 0, 10 and 25 µM CdSO4 and with addition of no, AB, TB or MDF biochar. It was proven that woody biochars mitigate Cd-induced effects in plants but no research has been executed concerning the mode-of-action. Few studies address the involvement of the phytohormone ethylene, although its concrete role in the biochar mechanism has yet to be discovered. Ethylene is important in several plant responses and is produced upon exposure to Cd. Ethylene is formed by the conversion of S-adenosylmethionine (SAM) to 1-aminocyclopropane-1-carboxylic acid (ACC) (rate-limiting step) by ACC synthase (ACS). ACS2 and ACS6 are the most important isoforms for Cd-induced ethylene production. For ethylene signalling in the plant, the EIN2 protein is important for signal transfer from the endoplasmic reticulum to the nucleus. An ethylene biosynthesis and signalling mutant were used to study the role of ethylene in the biochar mechanism (Chapter 5). The acs2-1acs6-1 mutant has a reduced Cd-induced ethylene production, whereas the ein2-1 mutant is insensitive to ethylene. Wild-type and ethylene mutant seedlings were cultivated via the SAFETY96 cultivation system. Immediately after sowing, seedlings were exposed to 0, 10 and 25 µM CdSO4 and no, AB, TB or MDF biochar. This study indicated that ethylene co-regulates the growth-enhancing potential of woody biochar in Cd-exposed A. thaliana seedlings. In conclusion, this study introduces a new screening method as well as the use of flow cytometric indices. Aquired data proved that the Cd-induced growth/defence trade-off is variable among plant parts and ages. This variety is rapidly screened by the use of SAFETY96 grown seedlings. Furthermore, our results indicate that woody biochars are able to mitigate the Cd-induced reduction of plant growth. More specific, woody biochars are able to reduce the Cd phytotoxicity by immobilisation of the metal. The negative effects of Cd on the regulation of the ROS level and cell cycle is mitigated by the woody biochars and this for both seedlings and more mature plants. Ethylene proves to play a role in this biochar mechanism since this phytohormone partly defines the degree of mitigation of Cd induced effects. Further research needs to establish the working model of ethylene in the woody biochar mechanism in A. thaliana seedlings.