Study of natural variation for Zn deficiency tolerance in Arabidopsis thaliana

English summary Zinc is an important structural component and co-factor of proteins in all living organisms. The model plant species for genetic and molecular studies, Arabidopsis thaliana, expresses more than 2,000 proteins with one or more Zn binding domains. Low Zn availability in arable soils is a widespread problem around the world which results in agricultural losses and the production of grains with low Zn content. The long-term consumption of low-Zn-content food items leads to severe health problems in humans as a result of severe or mild dietary Zn deficiency. Hence the importance of studying Zn homeostasis in plants and mechanisms involved in Zn deficiency tolerance aiming to enhance Zn concentration in plants edible parts and to develop varieties with a higher tolerance to Zn deficiency. Plants are sessile organisms which trough evolution have developed specific traits in order to adapt to certain environmental conditions in their surroundings. As a result some plant genotypes are more tolerant to Zn deficiency and when exposed to low Zn conditions are able to perform better than others. To investigate the physiological mechanisms involved in Zn deficiency tolerance I examined natural variation present in a set of twenty diverse Arabidopsis thaliana accessions. In chapter 2, differences in shoot biomass production, Zn usage index (ZnUI), ionome (concentration of elements) and expression level of six key Zn deficiency responsive genes were studied. Accessions did not show large natural variation for shoot Zn concentration under Zn deficiency, while the decreases in shoot biomass and ZnUI were more variable. The conclusion from this is that accessions differ for the minimum Zn concentration required for growth which is associated with differences in Zn deficiency tolerance. We also found that the gene expression levels of three Zn transmembrane transporters (IRT3, ZIP3 and 4) in shoot were positively correlated with ZnUI and shoot biomass, but negatively correlated with shoot Zn concentration. This implies that a higher tolerance to Zn deficiency in A. thaliana is associated with an increased Zn translocation from root to shoot under low Zn. Furthermore, I used a logistic regression model to demonstrate that differences in the shoot ionome can be used as a biomarker to identify the plant Zn physiological state. Based on the changes in the concentrations of some elements in each of the Zn deficiency treatments it was possible to predict the Zn physiological state of the plants similarly to when Zn concentration is used alone. The adaptive response to Zn deficiency involves physiological changes in shoots, but also in roots which play a key role in the acquisition of nutrients. In chapter 3 I used the same twenty A. thaliana accessions as described in chapter 2 to identify root system architecture traits and changes in the root ionome involved in a higher tolerance to Zn deficiency in plants. Similar to shoots, all accessions showed a strong reduction in root Zn concentration under Zn deficiency, whereas changes in other root system architecture traits were more variable between the accessions. These analyses showed that differences between the accessions in root system architecture traits and minimum Zn concentration required for growth are important for Zn deficiency tolerance. The Zn deficiency treatment also affects the formation of lateral roots and thus root system architecture. It was therefore not surprising that the Zn deficiency treatment induced changes in the concentrations of other elements which were correlated with changes in root traits. Plants respond to different concentrations of Zn supply by changing the expression levels of genes involved in the Zn homeostasis network. This is important for the control of the Zn concentration and sequestration in plant cells, tissues and organs and involves the uptake, accumulation, transport and redistribution of Zn within the plant. Based on the work described in chapter 2, three A. thaliana accessions were selected with contrasting tolerance to Zn deficiency, and used for a whole genome transcription profiling analysis using RNA sequencing. Chapter 4 describes the identification of sets of general and core genes used by A. thaliana in its response to Zn deficiency. The purpose of using three accessions was to complement previous studies, which used only one accession, and identify new candidate genes involved in the general response to Zn deficiency in A. thaliana. General transcriptional changes were observed in the regulation of carbohydrate metabolism, glucosinolate biosynthesis and the circadian clock. As the transcriptional changes were recorded at two time points, it was also possible to distinguish early and late responses to Zn deficiency. The early response to Zn deficiency was stronger in roots with the induction of several Zn homeostasis genes and repression of Fe uptake genes. The late response to Zn deficiency comprised of the strong induction of several Zn uptake, transport and remobilization genes in both roots and shoots. These analysis confirmed several genes previously identified in Col-0 to have a general role in the Zn deficiency response, but it also led to the identification of new candidate genes, such as defensins and defensin-like genes, as very promising new actors in the A. thaliana Zn deficiency homeostasis network. Chapter 5 describes the A. thaliana accession-specific Zn deficiency responsive transcript profiles, comparing Tsu-0, Pa-2 and Col-0, with the aim to identify biological processes involved in the observed differences in Zn deficiency tolerance between these three accessions. Tsu-0 displayed a high tolerance to Zn deficiency in shoot, Col-0 (reference accession) showed a high tolerance to Zn deficiency in both root and shoot, whereas Pa-2 root and shoot were more sensitive to Zn deficiency. Some of the accession-specific Zn deficiency responsive transcripts were involved in similar biological processes, such as defence response, programmed cell death and carbohydrates and glucosinolates metabolism. The differential regulation of these processes between the three accessions may reflect their differences in Zn deficiency tolerance. Among the Col-0 specific transcripts were several genes encoding proteins kinases which may play a role in a more specific separation of the abiotic and biotic stress responses in this accession and possibly involved in its higher tolerance to Zn deficiency in both shoots and roots. Tsu-0 specifically changes the expression of a set of shoot transcripts encoding ethylene responsive transcription factors which are involved in the regulation of shoot growth and plant tolerance to abiotic and biotic stresses, corresponding well with the observed shoot Zn deficiency tolerance. Accession Pa-2 down-regulated transcripts involved in cell wall organization in roots which correlates with its high sensitivity to Zn deficiency in this organ. Finally, the accessions specific response to Zn deficiency also resulted in the differential regulation of transcripts encoding transposases which may reflect large scale chromatin reorganization or demethylation in response to the stress condition. The main findings of the research described in this thesis and their implications are described in the General Discussion (chapter 6). By investigating the response to Zn deficiency in a diverse set of A. thaliana accessions both at the physiological and transcriptional level important mechanisms involved in Zn deficiency tolerance were identified. Furthermore, several key candidate genes among the accessions general and accession-specific Zn deficiency responsive transcripts were identified. The further functional characterization of these genes is expected to reveal important new steps in the regulation of Zn homeostasis and Zn deficiency tolerance in A. thaliana.