Browsing by Author "Ndah, Renata"

Aluminum (Al3+) toxicity is a major limiting factor in acidic soils when pH<5.5 and faba bean experiences yield decreases in these conditions. The multidrug and toxic compound extrusion (MATE) family plays a vital role in Al3+ tolerance across species. This study searched for an ortholog of MtMATE66, a gene involved in Al3+ resistance in barrel medic, in faba bean and quantified the ortholog’s relative expression in 4 faba bean genotypes (GPID_0022, GPID_0153, GPID_0178 and GPID_0191). pBLAST of MtMATE66 in the faba bean unpublished genome identified the ortholog jg20333.t1, with 88.6 % identity, e-value 0.0 and bit score 880. InterPro Scan and NCBI CDD conserved domain queries classified jg20333.t1 as a MATE in the DinF subfamily. MEME Suite identified the 50-amino acid citrate exuding motif characteristic to MATEs exuding citrate in Al3+ tolerance while a multiple sequence alignment and phylogenetic analysis clustered jg20333.t1 with Al3+ -tolerant citrate exuding MATEs. The SWISS Model 3D structure and DeepTMHMM predicted an α-helical, twelve-transmembrane protein topology. The plasma membrane was predicted as the subcellular localisation of jg20333.t1 by ProtComp, WoLFSPORT and YLoc, however SignalP identified no signal peptides. The molecular weight 54.57kDa, theoretical isoelectric point 8.60 and grand average of hydropathicity 0.67 of jg20333.t1 were calculated by ProtParam. EMBOSS Needle and GSDS aligned 13 exons. Four biological replicates of faba bean plants were set up for 3 different treatments: acidic of pH 3.81 (Ac), acidic of pH 3.81 plus Al3+ (Al) and neutral of pH 6.01 (N) in a greenhouse and root tip samples were collected 45 days after transplanting in peat media for RNA extraction. The relative expression of jg20333.t1 was determined by RT-qPCR of jg20333.t1 as target gene, Vfactin as reference gene and N as internal calibrator. The Cq values generated were analysed using the 2-ΔΔCq method and showed high relative fold change in both Ac and Al. The upregulation in Al confirmed the implication of jg20333.t1 in faba bean tolerance to Al3+. The upregulation in Ac suggests upstream regulation by STOP transcription factor. The four genotypes had no significant difference in fold change. Based on these results, it is concluded that jg20333.t1 is a faba bean MATE gene, VfMATE, implicated in Al3+ tolerance by citrate exudation.