Lipid Remodeling Confers Osmotic Stress Tolerance to Embryogenic Cells during Cryopreservation

Abstract

Plant species conservation through cryopreservation using plant vitrification solutions (PVS) is based in empiricism and the mechanisms that confer cell integrity are not well understood. Using ESI-MS/MS analysis and quantification, we generated 12 comparative lipidomics datasets for membranes of embryogenic cells (ECs) of Magnolia officinalis during cryogenic treatments. Each step of the complex PVS-based cryoprotocol had a profoundly different impact on membrane lipid composition. Loading treatment (osmoprotection) remodeled the cell membrane by lipid turnover, between increased phosphatidic acid (PA) and phosphatidylglycerol (PG) and decreased phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The PA increase likely serves as an intermediate for adjustments in lipid metabolism to desiccation stress. Following PVS treatment, lipid levels increased, including PC and PE, and this effectively counteracted the potential for massive loss of lipid species when cryopreservation was implemented in the absence of cryoprotection. The present detailed cryobiotechnology findings suggest that the remodeling of membrane lipids and attenuation of lipid degradation are critical for the successful use of PVS. As lipid metabolism and composition varies with species, these new insights provide a framework for technology development for the preservation of other species at increasing risk of extinction.

Keywords: Magnoliaceae; cryopreservation; cryoprotectant agents; ex situ conservation; lipid; plant vitrification solutions.

Figure 1

Steps in the cryopreservation process for embryogenic cell aggregates (ECs) of Magnolia officinalis. (I) Control (normal growth) (C); (II) loading treatment (osmoprotection) for 20 min at room temperature (L); (III) plant vitrification solution 2 (PVS2) treatment (dehydration with vitrification solution) for 15 min at 0 °C (P); (IV) freezing in liquid nitrogen (rapid cooling) for 1 day (F); (V); thawing (rapid warming) at 40 °C for 2 min (T); (VI) unloading treatment (rehydration and dilution of the vitrification solution) for 20 min at room temperature (U); and (VII) recovery growth for 2 weeks in normal growth conditions (R). To separate out the effects of individual steps in the overall cryopreservation process, single steps were excluded from the protocol (Treatments 1–6). Samples were also taken from six other condition combinations (Treatments 7–12) but with one step missing: (−L) no loading treatment; (−L+P) no loading treatment, but extended PVS2 treatment time (35 min); (−P) no PVS2 treatment; (−P+L) no PVS2 treatment, but extended loading treatment (35 min); (−FT) no freezing and thawing; and (−U) no unloading treatment.

Figure 2

Viability counts of Magnolia officinalis embryogenic cells (ECs) during standard- and altered-protocol cryopreservation. (a) Viable ECs show green fluorescence after fluorescein diacetate (FDA) staining, while dead tissues remain unstained. (b) Photographs are representative of three independent replicates in each case. Fluorescence intensities were recorded, and values are mean ± SD (n = 3). Explanations of the notations used for each step are provided in full in the legend to Figure 1. Columns with different letters are significantly different (p < 0.05).

Figure 3

Effect of cryopreservation processes on the phospholipid profile of Magnolia officinalis embryogenic cell (ECs). Left panel: Standard-protocol cryopreservation. Right panel: Altered-protocol cryopreservation. Each colored bar within a column represents a lipid species in the indicated treatments (see Figure 1 legend for notation). The color of each bar represents the level of the corresponding lipid species (nmol/mg dry weight). A total of 113 lipid species in the indicated lipid classes are organized using class (as indicated), total acyl carbons (in ascending order within a class), and total double bonds (in ascending order within a class and total acyl carbons). The dry weight is the dry weight of tissue after lipid extraction.

Figure 4

Molecular percentages of lipid classes in Magnolia officinalis ECs after cryopreservation treatments. Twelve different treatments (see Figure 1 legend for notation) were examined and compared. Values are means of n = 5.

Figure 5

Changes in levels of eight phospholipids in Magnolia officinalis ECs during cryopreservation. The fold changes were calculated using the average amount of total lipids of each class (n = 5) with the formula (A2 − A1)/A1, where A2 is the total lipid class amount for the indicated process (notation in Figure 1 legend) and A1 is the total lipid amount in the control.

Figure 6

Changes in levels (nmol/mg) of the molecular species of galactolipids and phospholipids during various Magnolia officinalis EC cryopreservation protocols. (a) Standard-protocol cryopreservation steps. (b) Altered-protocol cryopreservation. See Figure 1 legend for treatment notation. Values are mean ± SD (n = 5).

Figure 7

Schematic representation of membrane composition changes in Magnolia officinalis ECs during cryopreservation. The columns represent phosphatidylcholine (PC) and phosphatidylethanolamine (PE), while the triangles represent phosphatidic acid (PA). The number of columns and triangles represents the total amount of lipids.