Evolution, and functional analysis of Natural Resistance-Associated Macrophage Proteins (NRAMPs) from Theobroma cacao and their role in cadmium accumulation

Abstract

The presence of the toxic metal cadmium (Cd2+) in certain foodstuffs is recognised as a global problem, and there is increasing legislative pressure to reduce the content of Cd in food. The present study was conducted on cacao (Theobroma cacao), the source of chocolate, and one of the crops known to accumulate Cd in certain conditions. There are a range of possible genetic and agronomic methods being tested as a route to such reduction. As part of a gene-based approach, we focused on the Natural Resistance-Associated Macrophage Proteins (NRAMPS), a family of proton/metal transporter proteins that are evolutionarily conserved across all species from bacteria to humans. The plant NRAMP gene family are of particular importance as they are responsible for uptake of the nutritionally vital divalent cations Fe2+, Mn2+, Zn2+, as well as Cd2+. We identified the five NRAMP genes in cacao, sequenced these genes and studied their expression in various organs. We then confirmed the expression patterns in response to variation in nutrient cation availability and addition of Cd2+. Functional analysis by expression in yeast provided evidence that NRAMP5 encoded a protein capable of Cd2+ transport, and suggested this gene as a target for genetic selection/modification.

Cadmium isotope fractionation in the soil – cacao systems of Ecuador: a pilot field study

“…In summary, these results suggest that further, more comprehensive, coupled Cd isotope and concentration studies should be carried out to conrm whether such analyses indeed provide useful constraints on the origin and cycling of Cd in soil cacao systems and the partitioning of Cd within cacao plants. In particular, the isotopic data may be of utility for molecular and genetic studies, 63 which intend to identify the transporter proteins that govern Cd accumulation and partitioning in cacao plants, as well as for breeding efforts and the development of farming practices that aim to reduce the relatively high Cd concentrations of cacao beans grown on Cd-rich soils.…”

Manganese in Plants: From Acquisition to Subcellular Allocation

“…Under Mn-deficient conditions, the expression of AtNRAMP1 is moderately up-regulated, and nramp1 knockout mutants accumulate less Mn in shoots under Mn deficiency, which points to a function of this protein as a high-affinity Mn 2+ uptake transporter (Cailliatte et al, 2010). NRAMP1 orthologs in cacao, rapeseed, and peanut are also expressed in roots and complement a Mn uptake-deficient yeast strain, smf1, lacking a Mn transporter located in the plasma membrane (Meng et al, 2017;Ullah et al, 2018;Wang et al, 2019). In addition, active Mn 2+ uptake may be accomplished by the ZIP transporter AtIRT1 (Castaings et al, 2016), that is considered as the major Fe uptake transporter of dicots.…”

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

“…Trichobel, respectively. Detailed protocol of RNA isolation, reverse transcription and gene cloning is described elsewhere 22 . Sequence data for these genes are available in the NCBI GenBank database as follows: TcNRAMP5 (accession number H615049), TcHMA2 (MT151685), TcHMA3 (MT151686), TcHMA3 SV (MT151687) and PtMt2b gene 45 (MN974475).…”

“…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 .…”