DOI: 10.5061/dryad.tb2rbp07w
title: cDNA sequence of E2 gene family in Arabidopsis thaliana and data of statistical analysis

description: E2 ubiquitin-conjugating enzymes act as a heart role in the ubiquitination
 process and are responsible for catalysis ubiquitin transfer. Although the
 function of ubiquitin-protein ligases (E3s) in plant response to diverse
 abiotic stress by targeting specific substrates has been well studied, the
 E2s' involvement in environmental responses and their downstream
 targets are not well understood. Here, we demonstrated that the E2
 ubiquitin-conjugating enzyme 18 (UBC18) regulates the stability of FREE1
 to modulate iron deficiency stress. UBC18 affects the ubiquitination of
 FREE1 and promotes its degradation, overexpression of UBC18 in plants
 decreases their sensitivity to iron deficiency by reducing the level of
 FREE1, and high accumulation of FREE1 in the ubc18 mutant resulted in
 sensitivity to iron deficiency. In addition, we demonstrated the lysine
 residues K227, K295, K315, and K540 are required for FREE1 ubiquitination
 and stability regulation, and mutation of these lysines of FREE1 residues
 resulted in sensitivity to iron starvation in plants. Taken together, our
 findings reveal a mechanism of UBC18 in response to iron deficiency stress
 by altering the abundance of FREE1, and further elucidate the role of
 ubiquitination sites in FREE1 stability regulation and the plant iron
 deficiency response.
description: # cDNA sequence of E2 gene family in Arabidopsis thaliana and data of
 statistical analysis 
 We uploaded the cDNA
 sequence of E2 gene family in *Arabidopsis thaliana* in a Word file, and
 the statistical analysis data used in "**UBC18 E2 conjugating enzyme
 destabilizes the ESCRT component FREE1 in plant iron deficiency
 responses**" are listed in supplementary table S2. * For cDNA
 sequence of E2 gene family, we use them to generate a phylogenetic tree,
 which is presented as Fig.S1 in the manuscript. * For the data of Fig 2 in
 Supplementary Table S2, it means the relative FREE1 protein level
 indicated in Fig.2F in the manuscript. we first use ImageJ software to
 analyze the His-Sumo-2HA-FREE1 protein abundance, and the protein level of
 0min in Col-0, *ubc18-3* and ubc18-2 were set 1 respectively. REP 1# and
 REP 2# represent two independent experiments. * For the data of Fig 3 in
 Supplementary Table S2, the data of Fig 3A in Supplementary Table S2
 represent the ubiquitination level of FREE1. We use ImageJ software to
 quantify the abundance of ubiquitinated FREE1 and the GFP-FREE1 protein
 abundance and analyze the relative percentage of ubiquitinated FREE1
 protein. * For the data of Fig 3 in Supplementary Table S2, the data of
 Fig 3B in Supplementary Table S2 represent the relative protein level of
 FREE1. We use ImageJ software to quantify the abundance of FREE1 protein
 and the tubulin protein abundance, and analysis the relative percentage of
 FREE1 protein in Col-0, *ubc18-3* and ubc18-2. * For the data of Fig 3 in
 Supplementary Table S2, the data of Fig 3D in Supplementary Table S2
 represent the relative root length. We use ImageJ software to quantify the
 root length of Col-0, *UBQ:GFP-FREE1*, *ubc18-3* and ubc18-2 seedlings
 indicated in Fig.3C in the manuscript, the root length data were
 quantified by GraphPad Prism software and presented in Fig.3D in the
 manuscript. * For the data of Fig 6 in Supplementary Table S2, the data of
 Fig 6C in Supplementary Table S2 represent the relative root length. We
 use ImageJ software to quantified the root length of Col-0,
 *UBQ:GFP-FREE14G#1*, *UBQ:GFP-FREE14G#2* indicated in Fig.6C in
 manuscript, the root length data were quantified by GraphPad Prism
 software and presented in Fig.6D in manuscript. * For the data of Fig S4
 in Supplementary Table S2, the data of Fig S4 in Supplementary Table S2
 represent the relative expression of *FREE1* gene in Col-0, *ubc18-3*
 plants. the data were quantified by GraphPad Prism software and presented
 in Fig.S4 in manuscript. * For the data of Fig S5 in Supplementary Table
 S2, the data of Fig S5 in Supplementary Table S2 represent the relative
 expression of *UBC18* gene in Col-0 plant. the data were quantified by
 GraphPad Prism software and presented in Fig.S5 in manuscript.