PhD Defense | Amber Mertens | The interaction of calcium and uranium during uranium stress in Arabidopsis thaliana: uptake, distribution and stress responses

The interaction of calcium and uranium during uranium stress in Arabidopsis thaliana: uptake, distribution and stress responses

Uranium (U) is a naturally occurring heavy metal, yet anthropogenic sources of U are omnipresent. Uranium mining, milling and the use of phosphate fertilizers contaminate many areas and ecosystems. Plants in these ecosystems exposed to U can encounter toxic effects. As such, U disturbs the nutrient balance, inhibits plants growth and induces oxidative stress. Uranium uptake and toxicity are largely dependent on its speciation. Uranium speciation can be influenced by environmental factors such as the pH of the nutrient solution and the presence of other elements for example calcium (Ca) in the nutrient solution. Previous research has suggested that Ca influences U uptake, while another study found that additional Ca in the nutrient medium alters U toxicity. However, further knowledge is needed to get an in-depth understanding of the influence of the interaction between Ca and U on both U uptake and toxicity. In this work, we integrated the effects of the external as well as the internal Ca levels on U uptake, distribution and toxicity in Arabidopsis thaliana.

First, we assessed the effects of external Ca levels on U uptake, distribution and toxicity in A. thaliana (Chapter 3). For this purpose, plants were grown in nutrient solutions with a low (30 µM), normal (300 µM) or high (3 mM) Ca concentration and then exposed to U (50 µM) for 24 or 72 h. The results showed a clear distinction in U response between roots and shoots. Whilst low Ca partially alleviated growth inhibition induced by U in roots, it did not influence oxidative stress levels or U uptake. A stronger growth inhibition was observed in U-exposed roots grown on medium with a high Ca concentration. On the contrary, in shoots, a low Ca concentration in the nutrient medium decreased the Ca shoot concentration, as well as the oxidative stress levels and growth inhibition after U exposure. The opposite was seen in U-exposed shoots supplied with a high Ca concentration in the nutrient solution. Since a relatively low amount of U (about 0.01%) reaches the shoots, the influence of Ca on the U-affected parameters (oxidative stress hallmark gene expression and shoot growth) in the shoots suggested that Ca signalling might be important in the U response in this organ. Furthermore, U exposure decreased the gene expression of Ca/H EXCHANGER (CAX) 1 in shoots and increased CAX3 and CAX4 expression in roots.

Therefore, in a next experiment, mutant plants deficient in one or more of these CAX transporters were exposed to U (25 or 50 µM) for 24 or 72 h. Again, a clear difference in involvement of the CAX transporters in roots versus shoots was observed. In roots (Chapter 4), it was suggested that CAX4 might play a role in regulating U transport in the root tissue towards the stele, without an influence of CAX1 or CAX4 on U toxicity. In contrast, in shoots (Chapter 5), CAX1 seemed to stimulate oxidative signalling, whereas a lower CAX4 expression resulted in higher shoot potassium levels. The results suggested that CAX transporters play a role in U responses in shoots, each in a different way.

Thirdly, to get a broader insight in the response of plants to U, the proteome (mass spectrometry) and transcriptome (RNA sequencing) of shoots exposed to U (25 µM, 72 h) were compared to those of control shoots (0 µM U). These results showed that in U-exposed shoots, besides oxidative stress levels, the antioxidative defence is also stimulated. Furthermore, the jasmonic acid (JA) biosynthesis enzymes were upregulated, supporting the previously suggested role for JA, a well-known stress-induced phytohormone in root-to-shoot signalling during U exposure. Several up- and downregulated processes also implied potential involvement of the target of rapamycin complex 1 (TORC1) in the growth inhibition that is observed during U exposure.

In conclusion, the results show the interplay of Ca (external and internal) and U in terms of U uptake, distribution and toxicity in A. thaliana and that this interaction manifests very differently in roots versus shoots. Furthermore, TORC1 might be involved in regulating the growth inhibition instigated by U. Also, signalling during U exposure involves Ca signalling (and potentially JA). The results of this work might contribute to a better understanding of the involvement of Ca and U in the responses of plants to U and aid in improving ways to clean up and/or safely utilize U-contaminated soils for example for the production of biofuel.

Promotor:

• An Cuypers (UHASSELT)

SCK CEN mentors:

• Nele Horemans (SCK CEN)