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

ABSTRACT: Sugarcane (Saccharum spp.) is one of the most important crops for sugar, biofuels and bioenergy production, becoming an important commodity in the world agricultural market for more than 100 countries. In this study, label-free quantitative proteomics and phosphoproteomics analyses were performed to investigate signaling events related to somatic embryo maturation and differentiation in sugarcane. Embryogenic callus (EC) at the multiplication (EC0) and after 14 days (EC14) under maturation were compared. A total of 251 phosphoproteins and 700 proteins were differentially regulated and accumulated, respectively, in the EC14/EC0 comparison. Metabolic pathways analysis showed that proteins and phosphoproteins are enriched in lysine degradation and starch/sucrose metabolism during multiplication, while differentiation of somatic embryo involves the regulation of energetic metabolism, including TCA cycle, oxidative phosphorylation, and carbon metabolism. Multiplication-related phosphoproteins were mainly associated to abscisic acid responses and transcriptional regulation comprising the TOPLESS (TPL), SNF1 kinase homolog 10 (KIN10), SEUSS (SEU), and LEUNIG_HOMOLOG (LUH) proteins. In maturation-related phosphoproteins, the phosphorylation of the proteins light harvesting complex photosystem ii, CURVATURE THYLAKOID 1B, vacuolar proton ATPase A1 and phytochrome interacting factor 3-LIKE 5 is associated to bioenergetic metabolism and carbon fixation. Motif analysis revealed 15 phosphorylation motifs, of which the [D-pS / T-x-D] motif was unique for the phosphopeptides identified during somatic embryo differentiation. Co-expression network analysis of proteins and phosphoproteins revealed the interaction between SNF1-related protein kinase 2 (SnRK2), abscisic acid responsive elements-binding factor 2 (ABF2), and KIN10, indicating the role of these proteins in the embryogenic competence in EC0. The interactions between ubiquitin-conjugating enzyme 5, ubiquitin-conjugating enzyme 35, small ubiquitin-like modifier 1, and histone deacetylase 1 may be involved in post-translational protein modification during the embryo maturation stage. argonaute 1 (AGO1) also interacts with POLTERGEIST (POL) and may integrate gene silencing and regulation of meristem identity during somatic embryo development. These results reveal novel dynamics of protein regulation in somatic embryogenesis and identify new potential players in somatic embryo differentiation and their phosphosites.

INSTRUMENT(S): timsTOF Pro, SYNAPT G2-Si

ORGANISM(S): Saccharum Hybrid Cultivar Sp80-3280

TISSUE(S): Plant Cell, Cell Culture

SUBMITTER: Vanildo Silveira

LAB HEAD: Vanildo Silveira

PROVIDER: PXD027388 | Pride | 2022-05-07

REPOSITORIES: Pride

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Project description:In somatic embryogenesis, embryos are formed from a single or small group of somatic cells in response to stress and hormonal stimuli. In this study, a high-throughput phosphoproteomic analysis was used to identify and quantify phosphoproteins related to the acquisition of embryogenic competence in sugarcane. Embryogenic (EC) and nonembryogenic (NEC) calli were compared at the multiplication phase, resulting in the identification of 1279 phosphoproteins. After the differential accumulation analysis, 163 phosphoproteins were identified as unique to EC, and nine were unique to NEC, while 51 were upaccumulated, and 40 were downaccumulated in EC compared to NEC. These phosphopeptides were mainly single phosphorylated peptides, with serine residues representing the major site of phosphorylation. The motif-x analysis revealed the enrichment of [xxxpSPxxx], [RxxpSxxx] and [xxxpSDxxx] motifs, which are predicted phosphorylation sites for several kinases related to stress responses. Gene ontology enrichment revealed an overrepresentation of biological processes involved in embryonic development, showing that EC-related phosphoproteins (unique to EC and upaccumulated) were associated with stress responses, regulation of gene expression and epigenetic modifications. The NEC-related phosphoproteins (unique to NEC and downaccumulated) were associated with the translation process. In this sense, EC-related phosphoproteins described as potential regulators of abiotic stress tolerance in response to ABA-induced signals such as OSK3 (a catalytic subunit of SnRK1), ABF1, LEAs, REM4.1 and RD29Bs were identified. On the other hand, the NEC-related phosphoproteins EDR1 and PP2Ac-2 were negative regulators of ABA, suggesting a role of ABA in the acquisition of embryogenic competence linked to stress tolerance. Moreover, EC-related phosphoproteins associated with epigenetic modifications, such as HDA6, HDA19, and TPL, as well as those involved in embryo development, including ASIL1, M3KE1, MCM2, and GRV2, were identified as putative potential regulators of embryogenic competence. Our results provide novel data that may help elucidate the phosphorylation mechanisms controlling the activity, affinity and localization of specific proteins during somatic embryogenesis and suggest a role for these proteins in the hormonal signalling cascade leading to stress adaptation by reprogramming genetic expression.

Project description:Sour passion fruit (Passiflora edulis Sims) has economic and social relevance and is an alternative crop mainly for family farming agriculture. The use of micropropagation by somatic embryogenesis offers scale-up clonal propagation with high phytosanitary quality. The aim of this work was to evaluate the influence of polyethylene glycol (PEG) on the maturation of somatic embryos associated with differential accumulation of proteins and changes in the endogenous polyamines (PAs) content during somatic embryogenesis of P. edulis ‘UENF Rio Dourado’. Maturation of somatic embryos was performed using embryogenic callus in MS culture medium with PEG 6% or without PEG (control). PEG 6% promoted the maturation of a significantly higher number of somatic embryos at globular and cotyledonary stages when compared to control treatment. The higher somatic embryo formation induced by PEG 6% was associated with an increase in endogenous contents of free spermine, a PA with an important role in the maturation process of somatic embryogenesis cultures. PEG also promoted a significantly higher content of putrescine and total free PAs at the end of maturation, which was relevant for the increase in the number of somatic embryos. Comparative proteomic analyses of PEG 6%/control revealed that PEG 6% treatment induced the up-accumulation of proteins related to the glycolytic process, generation of precursor metabolites energy, and response to light stimulus, we can highlight the enolase ENO1, triosephosphate isomerase (TPI), ribulose bisphophate carboxylase/oxygenase activase chloroplastic (RCA), glyceraldehyde-3-phosphate dehydrogenase GAPCP-1, chloroplastic (GAPCP-1), succinate dehydrogenase [ubiquinone] flavoprotein subunit1, mitochondrial (SDH1-1), pyruvate dehydrogenase E1 component subunit alpha, mitochondrial (IAR4), ATP synthase CF1 beta subunit (PB), malate dehydrogenase [NADP], chloroplastic (AT5G58330) and chlorophyll a-b binding protein 21, chloroplastic (LHB1B1), and the down-accumulated proteins related mainly to the cellular metabolic process with proteins such as NADP-dependent malic enzyme (NADP-ME4), phosphoenolpyruvate carboxylase 2 (PPC1), UDP-glucose 6-dehydrogenase 1 and 4 (UGD2) and sucrose synthase 2 isoform X2 (SSA) and cell division cycle protein 48 homolog (ATCDC48B). The use of PEG induced thematuration and development of somatic embryos of P. edulis Sims ‘UENF Rio Dourado’ by the differential accumulation of proteins and modulation of endogenous contents of PAs

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	2019_10_31_PEP_1.raw.zip	Raw
	2019_10_31_PEP_10.raw.zip	Raw
	2019_10_31_PEP_2.raw.zip	Raw
	2019_10_31_PEP_3.raw.zip	Raw
	2019_10_31_PEP_4.raw.zip	Raw

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

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