The Arabidopsis thaliana cat2 mutation is hidden by cadmium treatment and reveals an important role for CATALASE in root development

As sessile organisms, plants have developed intricate regulatory mechanisms to respond to environmental cues promptly and effectively. Under soil pollution, the root system is the primary plant organ system undergoing physiological and morphological changes to initiate a systemic response potentially leading to adaptation. Specifically, soil contamination with the heavy metal cadmium (Cd) stands out as a significant environmental stressor due to its non-biodegradability, bioaccumulation potential, and widespread presence in waters and soils as a pollutant. Among the various strategies that plants employ to cope with stressful conditions, such as modifications of hormonal levels, gene expression, and the production of signal molecules, the enhanced synthesis of antioxidant enzymes emerges as one of the most crucial defense systems. This occurs because, in most cases, the environmental stress is translated into oxidative/nitrosative stress within the plant. In this respect, catalase is a powerful antioxidant enzyme involved in the degradation of hydrogen peroxide (H2O2) into water and oxygen (Sharma and Ahmad, 2014). This enzyme is primarily found in peroxisomes, organelles that also play a role in synthesizing crucial signaling molecules, e.g., nitric oxide (NO) and in the biosynthetic steps of some plant hormones, e.g., indole-3-acetic acid (IAA) (Reumann and Bartel, 2016). In Arabidopsis thaliana, three catalase genes have been identified, CAT1, CAT2, and CAT3. CAT1 expression is predominant in pollen and seeds, CAT2 in photosynthetically active tissues, and CAT3 in vascular tissues and senescent leaves. Previous research has shown that while the deletion of CAT2 significantly reduces catalase activity by 90%, knockout mutations in CAT1 and CAT3 have minimal or negligible effects on catalase activity (Mhamdi et al., 2010). Given these findings, we decided to investigate the role of CAT2 gene in response to Cd exposure by growing A. thaliana CAT2 knockout mutant (cat2-1) (purchased by NASC and genetically screened for homozygosity) and the corresponding wild-type in in-vitro conditions for 10 days in the presence of 60µM CdSO4 (Brunetti et al., 2011). After the growth period, different analyses were conducted, focusing on the root system, including morpho-histological investigations and ROS and RNS content detection. The morphological analyses revealed that the cat2-1 plants had a shorter primary root and a reduced number of mature lateral roots compared to the wild-type. Preliminary histological analysis suggests that the reduced number of observed lateral roots may be attributed to a hindered development caused by an alteration in the cellular division pattern. Furthermore, the content of all the reactive oxygen/nitrogen species (ROS/RNS) examined, including superoxide anion (O2-), H2O2, NO, and peroxynitrite (ONOO-), increased in cat2-1 mutant, indicating a role of the enzyme on ROS and RNS homeostasis. Moreover, the increased H2O2 levels in mutant roots, relative to the wild-type, indicate that CAT2 might regulate intracellular H2O2 levels even in non-photosynthetic tissues as are those of the roots. Finally, the presence of Cd at the tested concentration hid most of the observed effects due to CAT2 knockout, suggesting that the role of the enzyme in mitigating oxidative/nitrosative damage resulting from the heavy metal exposure may be dependent on the intensity and duration of the stress.