Gain and loss of gene function shaped the nickel hyperaccumulation trait in Noccaea caerulescens

Abstract Nickel hyperaccumulation is an extreme adaptation to ultramafic soils observed in more than 500 plant species. However, our understanding of the molecular mechanisms underlying the evolution of this trait remains limited. To shed light on these mechanisms, we have generated a high-quality genome assembly of the metal hyperaccumulator Noccaea caerulescens. We then used this genome as reference to conduct comparative intraspecific and interspecific transcriptomic analyses using various accessions of N. caerulescens and the non-accumulating relative Microthlaspi perfoliatum to identify genes associated with nickel hyperaccumulation. Our results suggest a correlation between nickel hyperaccumulation and a decrease in the expression of genes involved in defense responses and the regulation of membrane trafficking. Surprisingly, these analyses did not reveal a significant enrichment of genes involved in the regulation of metal homeostasis. However, we found that the expression levels of selected metal transporter genes, namely, NcHMA3, NcHMA4, and NcIREG2, are consistently elevated in N. caerulescens accessions hyperaccumulating nickel. Furthermore, our analyses identified frameshift mutations in NcIRT1 associated with the loss of nickel hyperaccumulation in a few accessions. We further showed that the expression of a functional NcIRT1 in the roots of the La Calamine accession increases nickel accumulation in shoots. Our results demonstrate that NcIRT1 participates in nickel hyperaccumulation in N. caerulescens. They also suggest that nickel hyperaccumulation is an ancient trait in N. caerulescens that has evolved from the high and constitutive expression of several metal transporters, including NcIREG2, and that the trait was subsequently lost in a few accessions due to mutations in NcIRT1.