Cadmium and arsenic affect adventitious root formation and the definition of the quiescent centre in Arabidopsis thaliana (L.) Heynh plantlets

The heavy metal cadmium (Cd) and the semimetal arsenic (As) are the most toxic elements for animals and plants. Both are known to alter the architecture of the whole root system in numerous plants. In particular, in Arabidopsis thaliana, they reduce primary root (PR) growth, induce damages to the root anatomy and affect the root differentiation pattern (1-3). Adventitious roots (ARs) are essential for the post-embryonic development of the root apparatus in a wide range of plant species, contributing to anchorage, water-use efficiency, and extraction of nutrients from the soil. Moreover, their presence may enhance the plant ability to extract toxic elements from the soil. In A. thaliana, the quiescent centre (QC) of PR, lateral roots (LRs) and ARs controls the apical growth through an involvement of auxin and cytokinin (4,5). Therefore, the correct definition, maintenance and activity of the QC are essential for the organization of the roots. However, the effects of Cd and As on A. thaliana AR formation, and on QC definition, are unknown. The aim of this research was to investigate whether Cd and As affect AR formation in A. thaliana, and to determine their effects on QC definition in the ARs. To the aim, seeds of A. thaliana Columbia (Col) ecotype and of the transgenic line QC25::GUS (QC identity marker for PR, LRs and ARs; 5,6), were sown in vitro in the absence (control treatment) or in the presence of 60 μM CdSO4 (Cd), or 400 μM Na2HAsO4.7H2O, (As) or 60 μM CdSO4 plus 100 μM Na2HAsO4.7H2O (Cd+As). In order to favour AR formation, the exposure to light (long-days) was preceded by nine days under continuous darkness (16 days of total growth period). Mean length of PR, hypocotyl length and AR density were evaluated in the absence and presence of the pollutants in both wild type and QC25::GUS plantlets. Moreover, in QC25::GUS line, AR QC definition and maintenance were investigated through histochemical GUS assays. The presence of the GUS signal and its localization in the apical root meristem were investigated starting from the stage VII of AR development (i.e. the stage of QC definition, 5). The results show that both pollutants, alone or together, significantly reduced the PR and the hypocotyl growth, with no significant differences among treatments. The presence of 400 μM As reduced significantly the percentage of plantlets with ARs, compared to the control treatment, whereas Cd alone did not cause any reduction. However, the plantlets treated with Cd, alone or combined with As, showed a greater density of ARs, unlike those treated with only As, in which the AR density decreased significantly. Moreover, in As alone treatment, the most of the roots were primordia at early stages. In addition, the percentage of ARs without GUS-signal in the QC significantly increased in the presence of the pollutants, and especially with 400 μM As and the combined treatment, with respect to the control treatment. Cadmium alone also provoked a shift of the GUS-signal to the columella cells. All together, these results suggest that Cd and As differently affect the QC, resulting into different AR development. 1) P. Brunetti, L. Zanella, A. Proia, A. De Paolis, G. Falasca, M.M. Altamura, L. Sanità di Toppi, P. Costantino, M.Cardarelli (2011) J Exp Bot, 62, 5509-5519 2) A. Sofo, A. Vitti, M. Nuzzaci, G. Tataranni, A. Scopa, J. Vangronsveld, T. Remans, G. Falasca, M.M. Altamura, F. Degola, L. Sanità di Toppi (2013) Physiol Plant, 149, 487-498 3) J.M. Abercrombie, M.D. Halfhill, P. Ranjan, M.R. Rao, A.M. Saxton, J.S. Yuan, C.N. Stewart Jr (2008) BMC Plant Biol, 8, 87-101 4) K. Jiang, L.J. Feldman (2005) Annu Rev Cell Dev Biol, 21, 485-509 5) F. Della Rovere, L. Fattorini, S. D'Angeli, A. Veloccia, G. Falasca, M.M. Altamura (2013) Ann Bot 112, 1395-1407 6) S. Sabatini, D. Beis, H. Wolkenfelt, J. Murfett, T. Guilfoyle, J. Malamy, P. Benfey, O. Leyser, N. Bechtold, P. Weisbeek, B. Scheres (1999) Cell, 99, 463-472