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

Sugarcane (Saccharum spp.) is one of the most important crops for sugar, biofuel, and bioenergy production and has become an important commodity in the worldwide agricultural market in 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 multiplication (EC0) and after 14 days (EC14) of maturation were compared. The EC14/EC0 comparison found that 251 phosphoproteins and 700 proteins were differentially regulated and accumulated, respectively. Metabolic pathway analysis showed that these proteins and phosphoproteins were enriched in lysine degradation and starch/sucrose metabolism during multiplication, whereas the differentiation of somatic embryos was found to involve the regulation of energetic metabolism, including the TCA cycle, oxidative phosphorylation, and carbon metabolism. Multiplication-related phosphoproteins were mainly associated with abscisic acid responses and transcriptional regulation of the TOPLESS (TPL), SNF1 kinase homolog 10 (KIN10), SEUSS (SEU), and LEUNIG_HOMOLOG (LUH) proteins. Among the maturation-related phosphoproteins, the phosphorylation of light harvesting complex photosystem ii, CURVATURE THYLAKOID 1B, vacuolar proton ATPase A1 and phytochrome interacting factor 3-LIKE 5 was found to be associated with bioenergetic metabolism and carbon fixation. A motif analysis revealed 15 phosphorylation motifs, and among these, the [D-pS/T-x-D] motif was unique among the phosphopeptides identified during somatic embryo differentiation. A coexpression network analysis of proteins and phosphoproteins revealed interactions among SNF1-related protein kinase 2 (SnRK2), abscisic acid responsive elements-binding factor 2 (ABF2), and KIN10, which indicated the role of these proteins in embryogenic competence in EC0. The interactions among ubiquitin-conjugating enzyme 5, ubiquitin-conjugating enzyme 35, small ubiquitin-like modifier 1, and histone deacetylase 1 may be involved in posttranslational protein modification during embryo maturation. Argonaute 1 (AGO1) also interacts with POLTERGEIST (POL) and may integrate gene silencing with the 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.

Histone Deacetylase Inhibitors Increase the Embryogenic Potential and Alter the Expression of Embryogenesis-Related and HDAC-Encoding Genes in Grapevine (Vitis vinifera L., cv. Mencía)

The low induction rates of somatic embryogenesis are one of the main limitations in its routine application in the grapevine (Vitis vinifera L.). The use of an induction medium containing histone deacetylase inhibitors (trichostatin A and, mainly, sodium butyrate) resulted in an improvement of the embryogenic responses in grapevine (cv. Mencía) cotyledonary and recently germinated somatic embryos. The relative expression of several grapevine genes related to embryogenic competence or encoding histone deacetylase enzymes was studied in cotyledonary somatic embryos that were cultured in the presence of 0.5 mM sodium butyrate. The results showed a significant overexpression of the BBM and VvSERK2 genes after 24 h of culture, whereas the VvWOX2 gene was underexpressed less in treated versus untreated explants. The results suggest that the inhibitor may trigger a molecular response related to an increase in embryogenic competence and changes in the expression of associated genes. The treatment with sodium butyrate also produced significant variations in the expression of several histone deacetylase enzyme-encoding genes. These results may enhance the possibility of obtaining somatic embryos, reducing the seasonal constraints associated with the use of floral explants in grapevines.

AUXIN PULSE IN THE INDUCTION OF SOMATIC EMBRYOS OF Eucalyptus

ABSTRACT The objective of this study was to evaluate the effect of auxin pulse intervals on the induction of somatic embryos of Eucalyptus grandis x E. urophylla and to describe the embryogenic behavior of callus under the effect of auxinic stress. Cotyledons were inoculated in culture medium containing 207.07 µM picloram, a treatment considered as auxin pulse. Explants that were in the auxin pulse treatment were transferred to semisolid or liquid medium containing 20.71 µM picloram after one, two, four or eight days of auxin pulse. In a second experiment, explants that were on auxin pulse treatment were transferred to semi-solid medium containing 20.71 µM picloram after one, two or three days of auxin pulse. Auxiliary picloram pulse treatments (207.02 µM) can be used as an initial source of stress for the acquisition of embryogenic competence. The oxidation of cotyledonary explants may be considered as an indication of the formation of embryogenic calli. The presence of pectins in peripheral regions of somatic pro-embryos can be considered as a marker of somatic embryogenesis in cotyledonary explants of Eucalyptus grandis x E. urophylla.