Bryophyte Spores Tolerate High Desiccation Levels and Exposure to Cryogenic Temperatures but Contain Storage Lipids and Chlorophyll: Understanding the Essential Traits Needed for the Creation of Bryophyte Spore Banks

Abstract

Understanding the desiccation and freezing tolerance of bryophyte spores is vital to explain how plants conquered land and current species distribution patterns and help to develop efficient ex situ conservation methods. However, knowledge of these traits is scarce. We investigated tolerance to drying (at 15% relative humidity [RH] for two weeks) and freezing (1 h exposure to liquid nitrogen) on the spores of 12 bryophyte species (23 accessions) from the UK. The presence of storage lipids and their thermal fingerprint, and the levels of unfrozen water content, were determined by differential scanning calorimetry (DSC). The presence of chlorophyll in dry spores was detected by fluorescence microscopy. All species and accessions tested tolerated the drying and freezing levels studied. DSC suggested that 4.1−29.3% of the dry mass is storage lipids, with crystallization and melting temperatures peaking at around −30 °C. Unfrozen water content was determined <0.147 g H2O g−1 dry weight (DW). Most of the spores investigated showed the presence of chlorophyll in the cytoplasm by red autofluorescence. Bryophyte spores can be stored dry at low temperatures, such as orthodox seeds, supporting the creation of bryophyte spore banks. However, the presence of storage lipids and chlorophyll in the cytoplasm may reduce spore longevity during conventional storage at −20 °C. Alternatively, cryogenic spore storage is possible.

Keywords: chlorophyll; cryopreservation; desiccation tolerance; differential scanning calorimetry; ex situ conservation; in vitro germination; lipid crystallization; liquid nitrogen; unfrozen water content.

Figure 1

DSC melting (a) and cooling (b) scans of four bryophyte species (Bryum capillare acc. 2, Polytrichum formosum acc. 2, Orthodontium lineare acc. 3, Dicranum scoparium acc. 4). Black arrows indicate lipid melting transitions, usually occurring with two events in the melting scans (a): one around −80 or −90 °C (related to α crystals [46]) and one around −30 °C (related to β’ crystals [46]). White arrows indicate lipid crystallization transitions, usually occurring with three broad events around −90, −50 and −25 °C in the cooling scans (b), but also as a lipid recrystallization event around −60 or −70 °C in the warming scans (a). A dashed line was added to indicate the baseline of the scan considered. Asterisks indicate the broad second-order transition that marks the glass transition.

Figure 2

DSC melting (a) and cooling (b) scans of Polytrichum formosum acc. 1 spores containing the indicated water content. Samples were scanned at 10 °C min⁻¹ from 25 to −150 °C and then from −150 to 40 °C. Lipid melting transitions are expressed using black arrows for the melting events and white arrows for the crystallization events (as in Figure 1). When water content increased, other peaks appeared (blue arrows), increasing the enthalpy of the main event (∆Hm) and occurring at 0 °C in the melting scans (a). These peaks were attributed to water melting (a), and water + lipid crystallization (b).

Figure 3

Relationship between water content (g H₂O g⁻¹ DW) and enthalpy of melting transition (J g⁻¹ DW) measured for spores of 12 samples of Polytrichum formosum (acc. 1 and acc. 2) with diverse water content. The slope of the linear relationship of the two wettest samples (see equation in the figure panel) was used to calculate the ΔH of the water melting or freezing transition on a g⁻¹ H₂O basis. The intersection between this line and the horizontal line attributed to the TAG transitions shows the exact point below which melting or freezing transition cannot be observed [40].

Figure 4

Microscopy images of the spores of P. formosum (a,b), F. hygrometrica (c,d), A. undulatum (e,f) and B. capillare (g,h). (a,c,e,g) were taken by an optical microscope under white light. (b,d,f,h) were taken by epifluorescence after excitation at 450–480 nm (blue filter). Green plastids or chloroplasts are clearly visible in P. formosum (a), A. undulatum (e) and B. capillare (g), particularly due to the red autofluorescence emitted (b,f,h), respectively.