Proteome-Wide Analysis of Heat-Stress in Pinus radiata Somatic Embryos Reveals a Combined Response of Sugar Metabolism and Translational Regulation Mechanisms

Abstract

Somatic embryogenesis is the process by which bipolar structures with no vascular connection with the surrounding tissue are formed from a single or a group of vegetative cells, and in conifers it can be divided into five different steps: initiation, proliferation, maturation, germination and acclimatization. Somatic embryogenesis has long been used as a model to study the mechanisms regulating stress response in plants, and recent research carried out in our laboratory has demonstrated that high temperatures during initial stages of conifer somatic embryogenesis modify subsequent phases of the process, as well as the behavior of the resulting plants ex vitro. The development of high-throughput techniques has facilitated the study of the molecular response of plants to numerous stress factors. Proteomics offers a reliable image of the cell status and is known to be extremely susceptible to environmental changes. In this study, the proteome of radiata pine somatic embryos was analyzed by LC-MS after the application of high temperatures during initiation of embryonal masses [(23°C, control; 40°C (4 h); 60°C (5 min)]. At the same time, the content of specific soluble sugars and sugar alcohols was analyzed by HPLC. Results confirmed a significant decrease in the initiation rate of embryonal masses under 40°C treatments (from 44 to 30.5%) and an increasing tendency in the production of somatic embryos (from 121.87 to 170.83 somatic embryos per gram of embryogenic tissue). Besides, heat provoked a long-term readjustment of the protein synthesis machinery: a great number of structural constituents of ribosomes were increased under high temperatures, together with the down-regulation of the enzyme methionine-tRNA ligase. Heat led to higher contents of heat shock proteins and chaperones, transmembrane transport proteins, proteins related with post-transcriptional regulation (ARGONAUTE 1D) and enzymes involved in the synthesis of fatty acids, specific compatible sugars (myo-inositol) and cell-wall carbohydrates. On the other hand, the protein adenosylhomocysteinase and enzymes linked with the glycolytic pathway, nitrogen assimilation and oxidative stress response were found at lower levels.

Thermopriming-associated proteome and sugar content responses in Pinus radiata embryogenic tissue

“…The chromatographic separation was performed on a Triart C18 Capillary Column 1/32” (12 nm, S-3µm, 150 × 0.3 mm, YMC) and using a Triart C18 Capillary Guard Column (0.5 × 5 mm, 3 μm, 12nm, YMC). Further details of the LC methodology (mobile phases, flow rate etc) and the different mass spectrometer operation modes used for DDA and DIA experiments are resumed in Castander-Olarieta et al (2021a). The only differences are the following: LC program [5 – 30% of B (0 - 50 min), 30 – 98% of B (50 – 52 min), 98% of B (52-54 min), 98 - 5% of B (54 – 56min), and 5% of B (56 – 65 min], voltage of the ionization source (5500 V), nebulizer gas 1 (GS1) and gas 2 (GS2) pressures (25 and 10 psi respectively), candidate ions minimum count threshold (100) and mass spectrometer software version (Analyst® TF 1.8.1, Sciex®).…”

Transduction of Signals during Somatic Embryogenesis

“…Similarly, Touraev et al (1996) observed an increased number of tobacco somatic embryos from shoot tips following the imposition of high temperatures, ranging from 33 • C to 37 • C [80]. Recently, Castander-Olarieta et al (2021) reported that the exposure of Pinus radiata explants to 60 • C for 5 min significantly promotes embryogenic competence [81]. These effects were associated with an elevated abundance of proteins involved in the post-transcriptional regulation (ARGONAUTE 1D), as well as biosynthesis of fatty acids, sugars, and cell wall carbohydrates.…”

“…Programmed cell death (PCD) is a conserved process occurring during plant embryogenesis, which is responsible for the dismantling of the suspensor and the removal of subordinate embryos in gymnosperm seeds [81,82]. During in vitro embryogenesis PCD eliminates specific embryogenic cells [83], and it is required for shaping the embryo body, as unequivocally demonstrated in spruce [84].…”

Current Proteomic and Metabolomic Knowledge of Zygotic and Somatic Embryogenesis in Plants

“…Later, proteomics studies during partial desiccation of Picea asperata somatic embryos showed that stress-related proteins associated with osmosis, PGR metabolism, antioxidative proteins, defense proteins, and photosynthesis-related proteins were critical factors for improving the maturation and germination of somatic embryos [82]. Besides, high-temperature application during the initiation of embryonal masses of Pinus radiata exhibited improvement in the generation of the number of somatic embryos, which was associated with the over-accumulation of stress-related proteins and enzymes connected to the synthesis of fatty acids, myo-inositol, and cell-wall [83].…”