Establishment of a Landscape of UPL5-Ubiquitinated on Multiple Subcellular Components of Leaf Senescence Cell in Arabidopsis

Abstract

Catabolism of macromolecules is a major event in senescent cells, especially involving proteolysis of organelles and abnormally aggregated proteins, circulation of nutrients, and precise control of intracellular environmental balance. Proteasomes are distributed in the nucleus and cytoplasm; however, proteasomes in organelles are limited. In this study, multi-omics proteomic analyses of ubiquitinated proteins enriched by using antibody against “di-Gly-Lys” via a free labeling were used to investigate the global changes of protein levels and ubiquitination modification levels of upl5 mutant relative to wild-type plant; subcellular localization analysis of UPL5 was found to be located in the nucleus, cytoplasm, and plastid within the cell; and the direct lysine site patterns of UPL5 were screened by the H89R substitution in the tagged ubiquitinated assay. It suggests that UPL5 acting as a candidate of organelle E3 ligase either in the nucleus or cytoplasm or plastid modifies numerous targets related to nuclear transcription and plastid photosynthesis involving in Ca2+ and hormone signaling pathway in plant senescence and in response to (a)biotic stress protection.

RETRACTED: UPL5 modulates WHY2 protein distribution in a Kub-site dependent ubiquitination in response to [Ca2+]cyt-induced leaf senescence

“…Previous ubiquitomic analysis of the upl5 mutant relative to WT found that WHY2 protein was a candidate substrate for UPL5-mediated ubiquitination. 36 To further demonstrate this interaction, we first performed a yeast two-hybrid assay. UPL5-BD and WHY2-AD plasmids were co-transformed in yeast strain AH109, and the growth of the co-transformants on the deficient TRP/ADE/HIS/LEU medium indicated that UPL5 interacted with WHY2 in the yeast cell ( Figures 1 A and 1B).…”

“…Using tobacco leaf transient injection and Arabidopsis protoplast transformation, we showed that the full-length WHY2 interacted with UPL5 in mitochondria and the nucleus, the deleted mitochondrion transit peptide (mtp) version of WHY2 interacted with UPL5 in chloroplast, the deleted chloroplast transit peptide (ctp) version of WHY2 interacted with UPL5 in cytoplasm and nucleus as well as mitochondria ( Figures 1 D-1F), while single, full-length UPL5 fused GFP was detected in the nucleus, plastid, mitochondria, or cytoplasmic compartments. 36 , 37

Figure 1 Interaction of UPL5 and WHY2 colocalized in chloroplasts and nucleus, as well as mitochondria (A) Variants of WHY2 proteins in yeast two hybrid system. (B) Co-transformation of UPL5 and WHY2 variants in yeast cells was selected in medium deficient for Trpton (Trp), Hisdine (His), Adenine (Ade), and Leucine (Leu).

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“…Since plastid precursor proteins can be cleared from the cytosol by the UPS, it has been reported that the functioning of the E3 ubiquitin ligase Sp1 is dependent on changes in the abundance of TOC components. 38 To test whether UPL5 influences plastid precursor stability or plastid proteome assembly, we searched back our previous proteome dataset 36 and used immunodetection to observe whether UPL5 is involved in precursor stability and plastid proteome assembly. Notably, the protein abundance of CDC48A, OEP61, cpHSP70, cpHSP93/ClpC, and HSP90 were significantly upregulated.…”

“…NAD1, CCB382, SAG24, SAG29 , and SWEET7 ) was upregulated in the oeWHY2 and the upl5 plants but downregulated in the why2 and the oeUPL5 plants. Venn analyzing of DEPs of upl5 /WT 36 with dataset of SAGs (LSD3.0) showed that 23 SAGs was upregulated, and 16 SAGs was downregulated in the upl5 relative to WT ( Figures 5 G and S6 ). Therefore, modulation of UPL5-WHY2 affected plastid genome stability accompanied with leaf senescence.…”

“…Previous ubiquitomic analysis of a mutant of a HECT-type ubiquitin E3 ligase, upl5 compared to WT identified candidate UPL5-interacting partners and ubiquitination substrates, including WHY2. 36 This previous work was based on tandem affinity purification coupled with mass spectrometry. Here we use yeast two hybrid, bimolecular fluorescence complementation (BiFC) and coimmunoprecipitation (CoIP) assay in vivo and in vitro to demonstrate that UPL5, that interacts with WHY2 in the cytoplasm, plastid, and nucleus to modulate WHY2 distribution and protein abundance.…”