Ubiquitylomes and proteomes analyses provide a new interpretation of the molecular mechanisms of rice leaf senescence.

MAIN CONCLUSION: We identified a typical rice premature senescence leaf mutant 86 (psl86) and exhibited the first global ubiquitination data during rice leaf senescence. Premature leaf senescence affects the yield and quality of rice, causing irreparable agricultural economic losses. In this study, we reported a rice premature senescence leaf mutant 86 (psl86) in the population lines of rice (Oryza sativa) japonica cultivar 'Yunyin' (YY) mutagenized using ethyl methane sulfonate (EMS) treatment. Immunoblotting analysis revealed that a higher ubiquitination level in the psl86 mutant compared with YY. Thus, we performed the proteome and ubiquitylome analyses to identify the differential abundance proteins and ubiquitinated proteins (sites) related to leaf senescence. Among 885 quantified lysine ubiquitination (Kub) sites in 492 proteins, 116 sites in 94 proteins were classified as up-regulated targets and seven sites in six proteins were classified as down-regulated targets at a threshold of 1.5. Proteins with up-regulated Kub sites were mainly enriched in the carbon fixation in photosynthetic organisms, glycolysis/gluconeogenesis and the pentose phosphate pathway. Notably, 14 up-regulated Kub sites in 11 proteins were enriched in the carbon fixation in photosynthetic organism pathway, and seven proteins (rbcL, PGK, GAPA, FBA5, ALDP, CFBP1 and GGAT) were down-regulated, indicating this pathway is tightly regulated by ubiquitination during leaf senescence. To our knowledge, we present the first global data on ubiquitination during rice leaf senescence.

Single-Cell RNA Sequencing Efficiently Predicts Transcription Factor Targets in Plants.

Discovering transcription factor (TF) targets is necessary for the study of regulatory pathways, but it is hampered in plants by the lack of highly efficient predictive technology. This study is the first to establish a simple system for predicting TF targets in rice (Oryza sativa) leaf cells based on 10 × Genomics' single-cell RNA sequencing method. We effectively utilized the transient expression system to create the differential expression of a TF (OsNAC78) in each cell and sequenced all single cell transcriptomes. In total, 35 candidate targets having strong correlations with OsNAC78 expression were captured using expression profiles. Likewise, 78 potential differentially expressed genes were identified between clusters having the lowest and highest expression levels of OsNAC78. A gene overlapping analysis identified 19 genes as final candidate targets, and various assays indicated that Os01g0934800 and Os01g0949900 were OsNAC78 targets. Additionally, the cell profiles showed extremely similar expression trajectories between OsNAC78 and the two targets. The data presented here provide a high-resolution insight into predicting TF targets and offer a new application for single-cell RNA sequencing in plants.