Impairing both HMA4 homeologs is required for cadmium reduction in tobacco

Abstract

In tobacco, the heavy metal P1B-ATPases HMA4.1 and HMA4.2 function in root-to-shoot zinc and cadmium transport. We present greenhouse and field data that dissect the possibilities to impact the two homeologous genes in order to define the best strategy for leaf cadmium reduction. In a first step, both genes were silenced using an RNAi approach leading to >90% reduction of leaf cadmium content. To modulate HMA4 function more precisely, mutant HMA4.1 and HMA4.2 alleles of a Targeting Induced Local Lesions IN Genomes (TILLING) population were combined. As observed with RNAi plants, knockout of both homeologs decreased cadmium root-to-shoot transfer by >90%. Analysis of plants with segregating null and wild-type alleles of both homeologs showed that one functional HMA4 allele is sufficient to maintain wild-type cadmium levels. Plant development was affected in HMA4 RNAi and double knockout plants that included retarded growth, necrotic lesions, altered leaf morphology and increased water content. The combination of complete functional loss (nonsense mutation) in one homeologous HMA4 gene and the functional reduction in the other HMA4 gene (missense mutation) is proposed as strategy to limit cadmium leaf accumulation without developmental effects.

Cadmium isotope fractionation reveals genetic variation in Cd uptake and translocation by Theobroma cacao and role of natural resistance-associated macrophage protein 5 and heavy metal ATPase-family transporters

“…Such work has particularly focused on genes that encode either transporters, such as natural resistanceassociated macrophage proteins (NRAMPs) [18][19][20][21][22][23][24][25] , which mediate the entry of Cd from the rhizosphere, or those such as heavy metal ATPases (HMAs) [26][27][28][29][30][31][32] , which determine the deposition of Cd in vacuoles or its translocation in the plant. Significant findings include (i) that the level of NRAMP5 in rice may be linked to the higher uptake of Cd in this species than in maize 21 , (ii) combining knockout of one homeologous HMA4 gene (nonsense mutation) and reduction of the other (missense mutation) reduced Cd levels in tobacco, while maintaining plant vigour 27 , and (iii) the identification in wheat of HMA3 variants linked to reduced Cd accumulation 26 . This latter example led to the development of a kompetitive allelespecific polymerase chain reaction marker that is now used in a marker-assisted breeding programme 33 .…”

Improved Cd, Zn and Mn tolerance and reduced Cd accumulation in grains with wheat-based cell number regulator TaCNR2

“…In addition, cadmium (Cd), lead (Pb), mercury (Hg) and arsenic (As) are non-essential elements and affect plant growth. Cd is one of the most toxic pollutants in soil 10,11 , and it mainly derives from sewage sludge disposal, pesticides, fungicides and phosphorus-rich fertilizer use, such as industrial and agricultural production. Cd can seriously destroy the plant roots, as well as affect the normal growth and development of plants 12,13 .…”

di-Cysteine Residues of the Arabidopsis thaliana HMA4 C-Terminus Are Only Partially Required for Cadmium Transport

“…This remaining Cd(II)-binding capacity of AtHMA4CCAAc appeared to be sufficient, at low Cd(II) exposure, to at least partially enable Cd(II) transport and translocation to the plant shoot (Figure 1), whereas it was shown recently that the di-Cys motif of AtHMA4c is essential for Zn transport (Lekeux et al, 2018). In the past, several attempts have been made to identify mutations enabling metal transporters to discriminate between essential metals (e.g., Fe or Zn) and non-essential toxic metals (e.g., Cd) (Rogers et al, 2000;Pottier et al, 2015;Liedschulte et al, 2017), with the aim of favoring transport of specific metals for biofortification. For HMA4-related proteins in tobacco, modification of the protein sequence to reduce Cd accumulation in tobacco leaves resulted in drastically altered Zn homeostasis and consequently strongly impaired growth (Liedschulte et al, 2017).…”

“…In the past, several attempts have been made to identify mutations enabling metal transporters to discriminate between essential metals (e.g., Fe or Zn) and non-essential toxic metals (e.g., Cd) (Rogers et al, 2000;Pottier et al, 2015;Liedschulte et al, 2017), with the aim of favoring transport of specific metals for biofortification. For HMA4-related proteins in tobacco, modification of the protein sequence to reduce Cd accumulation in tobacco leaves resulted in drastically altered Zn homeostasis and consequently strongly impaired growth (Liedschulte et al, 2017). In contrast, plants expressing an AtHMA4 variant with a substitution of Phe 177 , present in transmembrane domain 2, into a Leu displayed significantly decreased Cd accumulation in shoots while Zn accumulation was maintained to a level enabling normal growth of Arabidopsis (Lekeux et al, 2019).…”