Secretome analysis revealed that cell wall remodeling and starch catabolism underlie the early stages of somatic embryogenesis in Pinus nigra

Abstract

Somatic embryogenesis is an efficient mean for rapid micropropagation and preservation of the germplasm of valuable coniferous trees. Little is known about how the composition of secretome tracks down the level of embryogenic capacity. Unlike embryogenic tissue on solid medium, suspension cell cultures enable the study of extracellular proteins secreted into a liquid cultivation medium, avoiding contamination from destructured cells. Here, we present proteomic data of the secretome of Pinus nigra cell lines with contrasting embryogenic capacity, accounting for variability between genotypes. Our results showed that cell wall-related and carbohydrate-acting proteins were the most differentially accumulated. Peroxidases, extensin, α-amylase, plant basic secretory family protein (BSP), and basic secretory protease (S) were more abundant in the medium from the lines with high embryogenic capacity. In contrast, the medium from the low embryogenic capacity cell lines contained a higher amount of polygalacturonases, hothead protein, and expansin, which are generally associated with cell wall loosening or softening. These results corroborated the microscopic findings in cell lines with low embryogenic capacity-long suspensor cells without proper assembly. Furthermore, proteomic data were subsequently validated by peroxidase and α-amylase activity assays, and hence, we conclude that both tested enzyme activities can be considered potential markers of high embryogenic capacity.

Keywords: amylase; black pine; contrasting embryogenic capacity; extracellular proteome; peroxidases; polygalacturonases.

Figure 1

Characterization of cell lines with high embryogenic capacity (HEC) and low embryogenic capacity (LEC). (A) HEC tissue on the proliferation medium. (B) LEC tissue on the proliferation medium. (C) Precotyledonary somatic embryos in HEC cell lines after 5 weeks of development on the maturation medium. (D) Cotyledonary somatic embryos in HEC cell lines after 8 weeks on maturation medium. (E) Bipolar somatic embryo observed in HEC tissue growing on the proliferation medium (e, embryonal part composed of tightly packed meristematic cells; s, suspensor composed of a bundle of long vacuolated cells). (F) Loosely organized meristematic cells (m) connected with long vacuolated suspensor cells without assembly into the bundle (v) observed in LEC tissue growing on the proliferation medium.

Figure 2

Visualization of the mathematical model in partial least square discriminant analysis (PLS-DA) of quantified proteins. HEC, high embryogenic capacity cell lines; LEC, low embryogenic capacity cell lines; A and B, biological replicates of a specific cell line.

Figure 3

Enzyme activities in culture media of cell lines with different embryogenic capacity. Mean values with the same letters are not significantly different according to Tukey’s test, p < 0.05, n = 3. Error bars indicate the standard error of the mean. Statistical analysis revealed a significant difference in enzyme activities between the low embryogenic capacity cell line (LEC) and high embryogenic capacity cell line (HEC) groups. (A) Peroxidase (POD) activity; LEC: E489, E490, and E483; HEC: E482, E486, and E494. (B) α-Amylase activity; LEC: E489, E490, and E483; HEC: E456, E477, and E494.

Figure 4

Schematic overview of proteins with altered accumulation in comparison between cell lines and their classification into groups by their biological function. The dashed line points to an alternative protein function. LEC, low embryogenic capacity cell lines; HEC, high embryogenic capacity cell lines; CHO, carbohydrates; AGPs, arabinogalactan proteins; CW, cell wall; EM, extracellular matrix.