Isotopically dimethyl labeling was applied in a quantitative post-translational modification (PTM) proteomic study of phosphoproteomic changes in the drought responses of two contrasting soybean cultivars. A total of 9457 phosphopeptides were identified subsequently, corresponding to 4571 phosphoprotein groups and 3889 leading phosphoproteins, which contained nine kinase families consisting of 279 kinases. These phosphoproteins contained a total of 8087 phosphosites, 6106 of which were newly identified and constituted 54% of the current soybean phosphosite repository. These phosphosites were converted into the highly conserved kinase docking sites by bioinformatics analysis, which predicted six kinase families that matched with those newly found nine kinase families. The overly post-translationally modified proteins (OPP) occupies 2.1% of these leading phosphoproteins. Most of these OPPs are photoreceptors, mRNA-, histone-, and phospholipid-binding proteins, as well as protein kinase/phosphatases. The subgroup population distribution of phosphoproteins over the number of phosphosites of phosphoproteins follows the exponential decay law, Y = 4.13e^−0.098X − 0.04. Out of 218 significantly regulated unique phosphopeptide groups, 188 phosphoproteins were regulated by the drought-tolerant cultivar under the water loss condition. These significantly regulated phosphoproteins (SRP) are mainly enriched in the biological functions of water transport and deprivation, methionine metabolic processes, photosynthesis/light reaction, and response to cadmium ion, osmotic stress, and ABA response. Seventeen and 15 SRPs are protein kinases/phosphatases and transcription factors, respectively. Bioinformatics analysis again revealed that three members of the calcium dependent protein kinase family (CAMK family), GmSRK2I, GmCIPK25, and GmAKINβ1 kinases, constitute a phosphor-relay-mediated signal transduction network, regulating ion channel activities and many nuclear events in this drought-tolerant cultivar, which presumably contributes to the development of the soybean drought tolerance under water deprivation process.

Wind is one of the most prevalent environmental forces entraining plants to develop various mechano-responses, collectively called thigmomorphogenesis. Largely unknown is how plants transduce these versatile wind force signals downstream to nuclear events and to the development of thigmomorphogenic phenotype or anemotropic response. To identify molecular components at the early steps of the wind force signaling, two mechanical signaling-related phosphoproteins, identified from our previous phosphoproteomic study of Arabidopsis touch response, mitogen-activated protein kinase kinase 1 (MKK1) and 2 (MKK2), were selected for performing in planta TurboID (ID)-based quantitative proximity-labeling (PL) proteomics. This quantitative biotinylproteomics was separately performed on MKK1-ID and MKK2-ID transgenic plants, respectively, using the genetically engineered TurboID biotin ligase expression transgenics as a universal control. This unique PTM proteomics successfully identified 11 and 71 MKK1 and MKK2 putative interactors, respectively. Biotin occupancy ratio (BOR) was found to be an alternative parameter to measure the extent of proximity and specificity between the proximal target proteins and the bait fusion protein. Bioinformatics analysis of these biotinylprotein data also found that TurboID biotin ligase favorably labels the loop region of target proteins. A WInd-Related Kinase 1 (WIRK1), previously known as rapidly accelerated fibrosarcoma (Raf)-like kinase 36 (RAF36), was found to be a putative common interactor for both MKK1 and MKK2 and preferentially interacts with MKK2. Further molecular biology studies of the Arabidopsis RAF36 kinase found that it plays a role in wind regulation of the touch-responsive TCH3 and CML38 gene expression and the phosphorylation of a touch-regulated PATL3 phosphoprotein. Measurement of leaf morphology and shoot gravitropic response of wirk1 (raf36) mutant revealed that the WIRK1 gene is involved in both wind-triggered rosette thigmomorphogenesis and gravitropism of Arabidopsis stems, suggesting that the WIRK1 (RAF36) protein probably functioning upstream of both MKK1 and MKK2 and that it may serve as the crosstalk point among multiple mechano-signal transduction pathways mediating both wind mechano-response and gravitropism.

To elucidate the early signaling components involved in thigmomorphogenesis in Arabidopsis thaliana, we combined microscopy and proximity-labeling (PL)-based quantitative biotinylproteomics to characterize the touch-responsive putative cytoskeleton-interacting protein WPRa4. Our findings revealed that WPRa4 localizes near plastids and interacts with cytosolic Plastid Movement-Impaired (PMI) proteins and a plastidic translocon component, suggesting a cytoskeleton-plastid network in mechanosensing. Bioinformatic analysis of PL and cross-linking mass spectrometry (XL-MS) data identified PMI4 as a key mediator, with pmi4 mutants lacking touch-induced bolting delay, rosette size reduction, and Ca²⁺ oscillations. Transcriptomics further showed that PMI4 regulates touch-responsive and jasmonic acid (JA)-associated genes, such as LOX2. We propose a molecular model where interconnected Cytoskeleton-Plastoskeleton Continuum (CPC) proteins act as early mechanosensors, integrating the touch responses of plant aerial organs with calcium signaling and transcriptional reprogramming in Arabidopsis.

Amino acid (AA) substitutions are pivotal modulators of cellular signaling and homeostasis. Current studies of AA substitutions rely mainly on genomic and transcriptomic evidence, while proteome-level characterization remains underexplored, even though such substitutions can arise beyond DNA and RNA mutations. Here we address this gap with PIPI-C, an open-search mass spectrometry tool, and establish an integrated AA substitutomics pipeline to profile AA substitutions across five cancer cohorts and dissect the regulatory roles of substitution-bearing proteins. By cross-referencing 13,399 identified AA substitutions with matched whole-exome sequencing data, we observed no overlap with patient somatic mutations. Specifically, 61% of these substitutions required two or more base alterations and could not be derived from single-nucleotide variants, rendering them exclusively detectable at the proteomic level. The remaining 39% of substitutions were compatible with single-base variations. PIPI-C captured these candidate substitutions at the proteome scale, consistent with a translational-stage origin as reported for alternate RNA decoding in mammals. We further identified notable substitutions, including F43S and E91D in hemoglobin subunit beta, P584T in filamin A, and A175N in fructose-bisphosphate aldolase B, and our pipeline provides a framework for generating hypotheses about AA substitutions in cancer escape.