Integrative proteomics and phosphoproteomics reveals phosphorylation networks involved in the maintenance and expression of embryogenic competence in sugarcane callus.

Plant embryogenic cell culture allows mass propagation and genetic manipulation, but the mechanisms that determine the fate of these totipotent cells in somatic embryos have not yet been elucidated. Here, we performed label-free quantitative proteomics and phosphoproteomics analyses to determine signaling events related to sugarcane somatic embryo differentiation, especially those related to protein phosphorylation. Embryogenic calli were compared at multiplication (EC0, dedifferentiated cells) and after 14 days of maturation (EC14, onset of embryo differentiation). Metabolic pathway analysis showed enriched lysine degradation and starch/sucrose metabolism proteins during multiplication, whereas the differentiation of somatic embryos was found to involve the enrichment of energy metabolism, including the TCA cycle and oxidative phosphorylation. Multiplication-related phosphoproteins were associated with transcriptional regulation, including SNF1 kinase homolog 10 (KIN10), SEUSS (SEU), and LEUNIG_HOMOLOG (LUH). The regulation of multiple light harvesting complex photosystem II proteins and phytochrome interacting factor 3-LIKE 5 were predicted to promote bioenergetic metabolism and carbon fixation during the maturation stage. A motif analysis revealed 15 phosphorylation motifs. The [D-pS/T-x-D] motif was overrepresented during somatic embryo differentiation. A protein-protein network analysis predicted interactions among SNF1-related protein kinase 2 (SnRK2), abscisic acid-responsive element-binding factor 2 (ABF2), and KIN10, which indicated the role of these proteins in embryogenic competence. The predicted interactions between TOPLESS (TPL) and histone deacetylase 19 (HD19) may be involved in posttranslational protein regulation during somatic embryo differentiation. These results reveal the protein regulation dynamics of somatic embryogenesis and new players in somatic embryo differentiation, including their predicted phosphorylation motifs and phosphosites.

Label-Free Quantitative Phosphoproteomics Reveals Signaling Dynamics Involved in Embryogenic Competence Acquisition in Sugarcane.

In this study, a label-free quantitative phosphoproteomic analysis was performed to identify and quantify signaling events related to the acquisition of embryogenic competence in sugarcane. Embryogenic and nonembryogenic calli were compared at the multiplication phase, resulting in the identification of 163 phosphoproteins unique to embryogenic calli, 9 unique to nonembryogenic calli, and 51 upregulated and 40 downregulated in embryogenic calli compared to nonembryogenic calli. Data are available via ProteomeXchange with identifier PXD018054. Motif-x analysis revealed the enrichment of [xxxpSPxxx], [RxxpSxxx], and [xxxpSDxxx] motifs, which are predicted phosphorylation sites for several kinases related to stress responses. The embryogenic-related phosphoproteins (those unique and upregulated in embryogenic calli) identified in the present study are related to abscisic acid-induced signaling and abiotic stress response; they include OSK3, ABF1, LEAs, and RD29Bs. On the other hand, the nonembryogenic-related phosphoproteins EDR1 and PP2Ac-2 are negative regulators of abscisic acid signaling, suggesting a relationship between phosphoproteins involved in the abscisic acid and stress responses in the acquisition of embryogenic competence. Moreover, embryogenic-related phosphoproteins associated with epigenetic modifications, such as HDA6, HDA19, and TOPLESS, and with RNA metabolism, including AGO1, DEAH5, SCL30, UB2C, and SR45, were identified to play potential roles in embryogenic competence. These results reveal novel phosphorylation sites for several proteins and identify potential candidate biomarkers for the acquisition of embryogenic competence in sugarcane.

Embryogenic Competence Acquisition in Sugar Cane Callus Is Associated with Differential H+-Pump Abundance and Activity.

Somatic embryogenesis is an important biological process in several plant species, including sugar cane. Proteomics approaches have shown that H+ pumps are differentially regulated during somatic embryogenesis; however, the relationship between H+ flux and embryogenic competence is still unclear. This work aimed to elucidate the association between extracellular H+ flux and somatic embryo maturation in sugar cane. We performed a microsomal proteomics analysis and analyzed changes in extracellular H+-flux and H+-pump (P-H+-ATPase, V-H+-ATPase, and H+-PPase) activity in embryogenic and non-embryogenic callus. A total of 657 proteins were identified, 16 of which were H+ pumps. We observed that P-H+-ATPase and H+-PPase were more abundant in embryogenic callus. Compared to non-embryogenic callus, embryogenic callus showed higher H+ influx, especially on maturation day 14, as well as higher H+-pump activity (mainly, P-H+-ATPase and H+-PPase activity). H+-PPase appears to be the major H+ pump in embryogenic callus during somatic embryo formation, functioning in both vacuole acidification and PPi homeostasis. These results provide evidence for an association between higher H+-pump protein abundance and, consequently, higher H+ flux and embryogenic competence acquisition in the callus of sugar cane, allowing for the optimization of the somatic embryo conversion process by modulating the activities of these H+ pumps.

Salt stress induces changes in the proteomic profile of micropropagated sugarcane shoots.

Salt stress is one of the most common stresses in agricultural regions worldwide. In particular, sugarcane is affected by salt stress conditions, and no sugarcane cultivar presently show high productivity accompanied by a tolerance to salt stress. Proteomic analysis allows elucidation of the important pathways involved in responses to various abiotic stresses at the biochemical and molecular levels. Thus, this study aimed to analyse the proteomic effects of salt stress in micropropagated shoots of two sugarcane cultivars (CB38-22 and RB855536) using a label-free proteomic approach. The mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD006075. The RB855536 cultivar is more tolerant to salt stress than CB38-22. A quantitative label-free shotgun proteomic analysis identified 1172 non-redundant proteins, and 1160 of these were observed in both cultivars in the presence or absence of NaCl. Compared with CB38-22, the RB855536 cultivar showed a greater abundance of proteins involved in non-enzymatic antioxidant mechanisms, ion transport, and photosynthesis. Some proteins, such as calcium-dependent protein kinase, photosystem I, phospholipase D, and glyceraldehyde-3-phosphate dehydrogenase, were more abundant in the RB855536 cultivar under salt stress. Our results provide new insights into the response of sugarcane to salt stress, and the changes in the abundance of these proteins might be important for the acquisition of ionic and osmotic homeostasis during exposure to salt stress.