A HemK class glutamine‐methyltransferase is involved in the termination of translation and essential for iron homeostasis in Arabidopsis

Abstract

Iron (Fe) transport and utilization are controlled by Fe-dependent transcriptional cascades. Many genes participate in these processes, transcriptionally controlled by Fe-status. Thorough knowledge of the translational check-points is lacking.We identified a non-response to Fe-deficiency1-1 (nrf1-1) mutant of Arabidopsis thaliana, which displayed a hypersensitive phenotype under Fe-deficient conditions. By mapping nrf1-1, we found that the AT3G13440 locus encoding a HemK methyltransferase is responsible for the phenotype. Analyses of ProUBQ10:NRF1 CDS overexpression nrf1-1 lines and a T-DNA insertion mutant nrf1-2, confirmed that loss-of-function of NRF1 results in enhanced Fe-starvation-sensitivity.NRF1 is required for the proper expression of the majority of Fe-deficiency-inducible (FDI) genes. The nrf1 mutants accumulated more polysomes in the roots, due to stalled ribosomes on several transcripts. Ribosome-footprint (RF) mapping revealed that ribosomes are stalled at a stop codon that amplified the stalling of trailing ribosomes. We detected higher RF levels in many FDI transcripts in nrf1-2.Our study demonstrates the requirement of NRF1 for an accurate termination of protein synthesis essential not only for a precise iron homeostasis, but also cellular ion balance. NRF1 is also important for normal growth and development. A check-point that fine-tunes peptide release in plants is uncovered.

Histone H3 lysine4 trimethylation‐regulated GRF11 expression is essential for the iron‐deficiency response in Arabidopsis thaliana

“…M3 generation of ethyl methanesulfonate (EMS) mutated seeds of Pro IRT1:LUC reporter plants (Kailasam et al ., 2018) were screened under Fe‐deficient (−Fe) media for inhibition of luciferase activity. Through secondary screening in the M3 generation, a mutant along with nrf1 (Kailasam et al ., 2020), named non‐response to Fe‐deficiency2 (nrf2) , was selected, backcrossed three times with reporter line and denoted as nrf2‐1 . After mapping, all experiments were conducted in nrf2‐1 plants.…”

“…F 2 segregating populations were then analyzed under Fe‐deficient conditions, and homozygous nrf2‐1 progenies were selected. The genomic DNA was isolated from the pool of 80 homozygous progenies by DNeasy Plant Mini Kit (Qiagen) and concentrated by Genomic DNA Clean and Concentrator Kit (Zymo Research, Irvine, CA, USA) as previously described (Kailasam et al ., 2020). Further, a 100 bp sequencing library was prepared and was then used for the whole‐genome sequencing (single read) using an Illumina HiSeqTM 2500 sequencer.…”

“…Total protein was extracted as previously described (Kailasam et al ., 2020) from liquid nitrogen‐ground tissues. Proteins were separated on Nu‐PAGE 4‐12% Bis‐Tris gels (Invitrogen) and were immunodetected using anti‐IRT1 (Shanmugam et al ., 2011) as primary antibody followed by mouse immunoglobulin G (IgG) as secondary antibody (KPL; Cat: 474‐1806).…”

“…Differential gene expression was studied according to (Kailasam et al, 2018(Kailasam et al, , 2020. Briefly, tissues were frozen and ground in liquid nitrogen.…”

The Methyltransferase HemK Regulates the Virulence and Nutrient Utilization of the Phytopathogenic Bacterium Xanthomonas citri Subsp. citri

“…HemK/PrmC-class methyltransferases are conserved from bacteria [ 21 ] and yeast [ 22 , 23 ] to humans [ 24 ], and are responsible for the methylation of glutamine at the N5 position in the conserved GGQ motif of release factors. A few reports have been published on the important biological functions of HemK in plants [ 25 , 26 , 27 ], yeast [ 22 , 23 ], and bacteria [ 28 , 29 , 30 ]. In Arabidopsis , NRF1, a HemK-class glutamine-methyltransferase involved in the termination of translation, is essential for cellular iron homeostasis and the plant’s normal growth [ 25 ].…”