Rice stripe virus (RSV) is one of the most devastating viral pathogens in rice and can also cause the general chlorosis symptom in Nicotiana benthamiana plants. The chloroplast changes associated with chlorosis symptom suggest that RSV interrupts normal chloroplast functions. Although the change of proteins of the whole cell or inside the chloroplast in response to RSV infection have been revealed by proteomics, the mechanisms resulted in chloroplast-related symptoms and the crucial factors remain to be elucidated. RSV infection caused the malformation of chloroplast structure and a global reduction of chloroplast membrane protein complexes in N. benthamiana plants. Here, both the protoplast proteome and the chloroplast proteome were acquired simultaneously upon RSV infection, and the proteins in each fraction were analyzed. In the protoplasts, 1128 proteins were identified, among which 494 proteins presented significant changes during RSV; meanwhile, 659 proteins were identified from the chloroplasts, and 279 of these chloroplast proteins presented significant change. According to the label-free LC–MS/MS data, 66 nucleus-encoded chloroplast-related proteins (ChRPs), which only reduced in chloroplast but not in the whole protoplast, were identified, indicating that these nuclear-encoded ChRPswere not transported to chloroplasts during RSV infection. Gene ontology (GO) enrichment analysis confirmed that RSV infection changed the biological process of protein targeting to chloroplast, where 3 crucial ChRPs (K4CSN4, K4CR23, and K4BXN9) were involved in the regulation of protein targeting into chloroplast. In addition to these 3 proteins, 41 among the 63 candidate proteins were characterized to have chloroplast transit peptides. These results indicated that RSV infection changed the biological process of protein targeting into chloroplast and the location of ChRPs through crucial protein factors, which illuminated a new layer of RSV–host interaction that might contribute to the symptom development.

Cold limits the growth and yield of maize in temperate regions, but the molecular mechanism of cold adaptation remains largely unexplored in maize. To identify early molecular events during cold shock, maize seedlings were treated under 4 °C for 30 min and 2 h, and analyzed at both the proteome and phosphoproteome levels. Over 8500 proteins and 19,300 phosphopeptides were quantified. About 660 and 620 proteins were cold responsive at protein abundance or site-specific phosphorylation levels, but only 65 proteins were shared between them. Functional enrichment analysis of cold-responsive proteins and phosphoproteins revealed that early cold response in maize is associated with photosynthesis light reaction, spliceosome, endocytosis, and defense response, consistent with similar studies in Arabidopsis. Thirty-two photosynthesis proteins were down-regulated at protein levels, and 48 spliceosome proteins were altered at site-specific phosphorylation levels. Thirty-one kinases and 33 transcriptional factors were cold responsive at protein, phosphopeptide, or site-specific phosphorylation levels. Our results showed that maize seedlings respond to cold shock rapidly, at both the proteome and phosphoproteome levels. This study provides a comprehensive landscape at the cold-responsive proteome and phosphoproteome in maize seedlings that can be a significant resource to understand how C₄ plants respond to a sudden temperature drop.

WUSCHEL (WUS) is a crucial transcription factor in regulating plant stem cell development, and its expression can also improve genetic transformation. However, the ectopic expression of WUS always causes pleiotropic effects during genetic transformation, making it important to understand the regulatory mechanisms underlying these phenomena. In our study, we found that the transient expression of the maize WUS ortholog ZmWus2 caused severe leaf necrosis in Nicotiana benthamiana. We performed transcriptomic and non-target metabolomic analyses on tobacco leaves during healthy to wilted states after ZmWus2 transient overexpression. Transcriptomic analysis revealed that ZmWus2 transformation caused active metabolism of inositol trisphosphate and glycerol-3-phosphate, while also upregulating plant hormone signaling and downregulating photosystem and protein folding pathways. Metabolomic analysis mainly identified changes in the synthesis of phenylpropanoid compounds and various lipid classes, including steroid synthesis. In addition, transcription factors such as ethylene-responsive factors (ERFs), the basic helix–loop–helix (bHLH) factors, and MYBs were found to be regulated by ZmWus2. By integrating these findings, we developed a WUS regulatory model that includes plant hormone accumulation, stress responses, lipid remodeling, and leaf necrosis. Our study sheds light on the mechanisms underlying WUS ectopic expression causing leaf necrosis and may inform the development of future genetic transformation strategies.

Fruit ripening in tomato (Solanum lycopersicum) has been extensively studied at the transcriptomic level. However, comprehensive profiling of the tomato fruit proteome and phosphoproteome remains limited. In this study, we performed large-scale proteome and phosphoproteome profiling of tomato (Ailsa Craig) fruits across five ripening stages using tandem mass tags (TMT)-based quantitative proteomics. Our analysis quantified over 8800 proteins and 20,000 high-confidence phosphorylation sites. Ripening-associated phosphorylation and dephosphorylation events were identified in diverse ripening regulators, including transcription factors, ethylene biosynthesis and signaling proteins, and epigenetic modifiers. Weighted gene co-expression network analysis (WGCNA) revealed a tetratricopeptide repeat protein, REDUCED CHLOROPLAST COVERAGE 1a (REC1a), as a key regulator of fruit ripening. Parallel reaction monitoring (PRM)-based targeted proteomic analysis validated the expression profiles of REC1a and its three phosphorylation sites. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated knockout of REC1a resulted in reduced lycopene accumulation and slower chlorophyll degradation, highlighting its role in the chloroplast-to-chromoplast transition, which is critical for fruit pigmentation during ripening. Quantitative proteomic analyses of rec1a mutants demonstrated reduced levels of Clp proteases and chaperones, proteins known to regulate plastid transitions. Additionally, co-immunoprecipitation and split-luciferase complementation assays revealed that REC1a interacts with the eukaryotic translation initiation factor subunits eIF2α and eIF2Bβ, suggesting its role in regulating protein synthesis during ripening. This study provides the most comprehensive quantitative proteome and phosphoproteome atlas of tomato fruits to date and identifies REC1a as a regulator of fruit ripening, offering new insights into the underlying molecular mechanisms.