Construction and analysis of a Noccaea caerulescens TILLING population

Metals such as Zn or Cd are toxic to plant and humans when they are exposed in high quantities through contaminated soil or food. Noccaea caerulescens, an extraordinary Zn/Cd/Ni hyperaccumulating species, is used as a model plant for metal hyperaccumulation and phytoremediation studies. Current reverse genetic techniques to generate mutants based on transgenesis is cumbersome due to the low transformation efficiency of this species. We aimed to establish a mutant library for functional genomics by a non-transgenic approach, to identify mutants with an altered mineral profiling, and to screen for mutations in bZIP19, a regulator of Zn homeostasis in N. caerulescens.

To generate the N. caerulescens mutant library, 3000 and 5000 seeds from two sister plants of a single-seed recurrent inbred descendant of the southern French accession Saint-Félix-de-Pallières (SF) were mutagenized respectively by 0.3 or 0.4% ethyl methane sulfonate (EMS). Two subpopulations of 5000 and 7000 M2 plants were obtained after 0.3 or 0.4% EMS treatment. The 0.4% EMS treatment population had a higher mutant frequency and was used for TILLING. A High Resolution Melting curve analysis (HRM) mutation screening platform was optimized and successfully applied to detect mutations for NcbZIP19, encoding a transcription factor controlling Zn homeostasis. Of four identified point mutations in NcbZIP19, two caused non-synonymous substitutions, however, these two mutations did not alter the ionome profile compared to the wild type. Forward screening of the 0.4% EMS treatment population by mineral concentration analysis (ionomics) in leaf material of each M2 plant revealed putative mutants affected in the concentration of one or more of the 20 trace elements tested. Several of the low-Zn mutants identified in the ionomic screen did not give progeny, illustrating the importance of Zn for the species. The mutant frequency of the population was evaluated based on an average of 2.3 knockout mutants per tested monogenic locus.

The 0.4% EMS treatment population is effectively mutagenized suitable for forward mutant screens and TILLING. Difficulties in seed production in low Zn mutants, obtained by both forward and reverse genetic approach, hampered further analysis of the nature of the low Zn phenotypes.