Accurately identifying DNA polymorphisms can bridge the gap between phenotypes and genotypes and is essential for molecular marker assisted genetic studies. Genome complexities, including large-scale structural variations, bring great challenges to bioinformatic analysis for obtaining high-confidence genomic variants, as sequence differences between non-allelic loci of two or more genomes can be misinterpreted as polymorphisms. It is important to correctly filter out artificial variants to avoid false genotyping or estimation of allele frequencies. Here, we present an efficient and effective framework, inGAP-family, to discover, filter, and visualize DNA polymorphisms and structural variants (SVs) from alignment of short reads. Applying this method to polymorphism detection on real datasets shows that elimination of artificial variants greatly facilitates the precise identification of meiotic recombination points as well as causal mutations in mutant genomes or quantitative trait loci. In addition, inGAP-family provides a user-friendly graphical interface for detecting polymorphisms and SVs, further evaluating predicted variants and identifying mutations related to genotypes. It is accessible at https://sourceforge.net/projects/ingap-family/.

Organogenesis in plants occurs across all stages of the life cycle. Although previous studies have identified many genes as important for either vegetative or reproductive development at the RNA level, global information on translational and post-translational levels remains limited. In this study, six Arabidopsis stages/organs were analyzed using quantitative proteomics and phosphoproteomics, identifying 2187 non-redundant proteins and evidence for 1194 phosphoproteins. Compared to the expression observed in cauline leaves, the expression of 1445, 1644, and 1377 proteins showed greater than 1.5-fold alterations in stage 1–9 flowers, stage 10–12 flowers, and open flowers, respectively. Among these, 294 phosphoproteins with 472 phosphorylation sites were newly uncovered, including 275 phosphoproteins showing differential expression patterns, providing molecular markers and possible candidates for functional studies. Proteins encoded by genes preferentially expressed in anther (15), meiocyte (4), or pollen (15) were enriched in reproductive organs, and mutants of two anther-preferentially expressed proteins, acos5 and mee48, showed obviously reduced male fertility with abnormally organized pollen exine. In addition, more phosphorylated proteins were identified in reproductive stages (1149) than in the vegetative organs (995). The floral organ-preferential phosphorylation of GRP17, CDC2/CDKA.1, and ATSK11 was confirmed with western blot analysis. Moreover, phosphorylation levels of CDPK6 and MAPK6 and their interacting proteins were elevated in reproductive tissues. Overall, our study yielded extensive data on protein expression and phosphorylation at six stages/organs and provides an important resource for future studies investigating the regulatory mechanisms governing plant development.

Faithful pollen wall formation requires tight control of both the amplitude and duration of tapetal transcriptional activity. However, how such dynamic restraint is achieved during anther development remains poorly understood. Here, we identify a phosphorylation-dependent regulatory module in which SBI-PP2A-C1/C5 constrains the functional activity window of the tapetal transcription factor bHLH089 to safeguard male fertility. PP2A-C1 and PP2A-C5 physically associate with bHLH089 and limit its transcriptional output by reducing bHLH089 phosphorylation and nuclear retention, thereby restricting its nuclear accumulation and effective activity. Genetic analyses reveal that precise PP2A-C1/C5 dosage is critical for pollen wall integrity, as both reduced PP2A-C function and PP2A-C1/C5 overexpression disrupt tapetal transcriptional homeostasis and compromise male fertility. Mechanistically, Ser28 and Ser58 function as conserved phosphorylation-dependent determinants of bHLH089 nuclear retention and are required for PP2A-C1/C5-sensitive regulation of bHLH089 activity. Together, these findings identify a phosphatase-mediated timing mechanism that integrates transcriptional feedback with post-translational control to constrain bHLH089-driven transcriptional amplification within a self-limiting tapetal activity window required for tapetal homeostasis, pollen wall formation, and male fertility.

Tomato (Solanum lycopersicum) is popular among consumers worldwide for its abundant nutritional value, and carotenoids are important metabolites in tomato with significant nutritional and physiological significance. Previous studies have demonstrated that AP2/ethylene response factor (ERF) transcription factors are key regulators involved in carotenoid biosynthesis. However, most studies have focused on ERF activators that positively regulate carotenoid biosynthesis, while the mechanisms by which Ethylene-responsive element binding factor-associated Amphiphilic Repression (EAR) motif-containing ERF repressors modulate this process remain largely unidentified. Here, we identified a AP2/ERF transcription factor SlERF.F2, which harbors EAR motifs and is co-expressed with key enzyme genes in the carotenoid biosynthesis. Although SlERF.F2 contains the EAR motif typically characterizing negative regulatory transcription factors, the LC-MS carotenoids profiling analysis revealed that SlERF.F2 overexpression significantly increased total carotenoid content in fruit, whereas SlERF.F2 knockout reduced the carotenoids level. Biochemical and molecular assays revealed that SlERF.F2-mediated transcriptional repression of the carotenoid metabolism β-carotene hydroxylase gene (SlBCH1) and the green shoulder formation suppressor gene (SlBEL2), which is dependent on the presence of its C-terminal EAR motif. SlERF.F2 suppresses the expression of the SlBCH1 and SlBEL2 genes, thereby reducing β-carotene degradation and promoting green shoulder formation in tomatoes, respectively. Through this negative-negative regulatory mechanism, SlERF.F2 achieves carotenoid accumulation in tomatoes. Furthermore, we found that SlERF.F2 interacts with the corepressor SlTPL2 via the C-terminal EAR motif, synergistically inhibiting the activity of the SlBCH1 gene. Collectively, our study unveils a dual-function regulatory module where SlERF.F2 integrates carotenoid metabolism and chloroplast development, offering a strategic target for enhancing tomato fruit quality.