Desiccation Tolerance in Chlorophyllous Fern Spores: Are Ecophysiological Features Related to Environmental Conditions?

Abstract

Fern spores of most species are desiccation tolerant (DT) and, in some cases, are photosynthetic at maturation, the so-called chlorophyllous spores (CS). The lifespan of CS in the dry state is very variable among species. The physiological, biochemical, and biophysical mechanisms underpinning this variability remain understudied and their interpretation from an ecophysiological approach virtually unexplored. In this study, we aimed at fulfilling this gap by assessing photochemical, hydric, and biophysical properties of CS from three temperate species with contrasting biological strategies and longevity in the dry state: Equisetum telmateia (spore maturation and release in spring, ultrashort lifespan), Osmunda regalis (spore maturation and release in summer, medium lifespan), Matteuccia struthiopteris (spore maturation and release in winter, medium-long lifespan). After subjection of CS to controlled drying treatments, results showed that the three species displayed different extents of DT. CS of E. telmateia rapidly lost viability after desiccation, while the other two withstood several dehydration-rehydration cycles without compromising viability. The extent of DT was in concordance with water availability in the sporulation season of each species. CS of O. regalis and M. struthiopteris carried out the characteristic quenching of chlorophyll fluorescence, widely displayed by other DT cryptogams during drying, and had higher tocopherol and proline contents. The turgor loss point of CS is also related to the extent of DT and to the sporulation season: lowest values were found in CS of M. struthiopteris and O. regalis. The hydrophobicity of spores in these two species was higher and probably related to the prevention of water absorption under unfavorable conditions. Molecular mobility, estimated by dynamic mechanical thermal analysis, confirmed an unstable glassy state in the spores of E. telmateia, directly related to the low DT, while the DT species entered in a stable glassy state when dried. Overall, our data revealed a DT syndrome related to the season of sporulation that was characterized by higher photoprotective potential, specific hydric properties, and lower molecular mobility in the dry state. Being unicellular haploid structures, CS represent not only a challenge for germplasm preservation (e.g., as these spores are prone to photooxidation) but also an excellent opportunity for studying mechanisms of DT in photosynthetic cells.

Keywords: desiccation tolerance; dynamic mechanical analysis; environmental conditions; glassy state; green spores; molecular mobility; tocopherol; water relations.

Figure 1

Water content (WC) (A), percentage of recovery of Fv/Fm (B), and percentage of germination (C) relative to the respective control values after first rehydration (R1) (gray bars) and second rehydration (R2) (white bars) at 10% RH in darkness in the spores of the three studied ferns. Control values of Fv/Fm in nontreated spores were obtained after 24 h of hydration in darkness. Control values of germination in nontreated spores were obtained 10 days after sowing on Dyer medium. Each bar represents the mean ± SE (n = 4). Different letters indicate significant differences among species and dehydration–rehydration (D–H) cycles (P < 0.05).

Figure 2

Percentage of Fo relative to the respective control values in the CS of the three studied ferns after desiccation (D1) (gray bars) and second desiccation (D2) (white bars). Control values were obtained as in Figure 1 . Each bar represents the mean ± SE (n = 4). Different letters indicate significant differences among species and dehydration–rehydration (D–R) cycles (P < 0.05).

Figure 3

Dynamic mechanical thermal analysis (DMTA) scans of ORe, MSt, and ETe CS desiccated at 10% RH for 48 h. α-Relaxation is characterized by a peak in the tan δ between 30 and 50°C (upper panel) that coincides with a large decrease in storage modulus (G’) (lower panel). α-Relaxation coincides in temperature with the glass transition temperature (Tg) measured by differential scanning calorimetry (Ballesteros et al., 2017). Other molecular relaxations are observed above and below the Tg that are associated to melting and gelation events (as from Ballesteros and Walters, 2011) and melting of the storage lipids (Ballesteros et al., 2017), respectively. One representative curve (from n = 3 independent biological replicates) is shown for each spore species.

Figure 4

Water potential (A) and water content (WC) (B) in the spores of the three studied ferns at full turgor, at turgor loss point, and in the sporangial state. Gray bars correspond to water potentials at full turgor (Ψ_O) (left axis), white bars correspond to water potential at turgor loss point (TLP) (Ψ_TLP), and black bars correspond to water potential in the sporangial state (Ψ_SPO) (right axis). The same color code is used for water content (WC). Each bar represents the mean ± SE (n = 3). Different letters indicate significant differences among species for the same parameter (P < 0.05).

Figure 5

Static contact angles of 6 µl distilled water drops over the spores of the three studied species in their sporangial state. (A) Time course of the change in the contact angle during 10 s. Color lines represent the average ± SE (n = 10) for each of the three species evaluated MSt (orange line), ORe (blue line), and ETe (green line). Contact angles at selected time points during the recording: 1 s (black bars), 5 s (gray bars), and 10 s (white bars) after water drop contacted with the spore surface (B). Each bar represents the mean ± SE (n = 10). Different letters indicate significant differences among species and time points (P < 0.05). Details of a representative distilled water drop in contact with spores surface at time 1 s are shown as insets.

Figure 6

SEM micrographs of spores of ORe (A), MSt (B), and ETe (C) dried in silica gel during 24 h. The scale of each micrograph is indicated in the lower part. Abbreviations: (BP), broken perispore (E),elaters; (F), folds; U undulations.

Figure 7

Summary view of the main trends on the physiological and physicochemical features in CS of the three ferns studied. Arrows indicate higher or lower values when each parameter is compared among species.