Outstanding Ultra-Low Freezing Tolerance in Moss Species: Insights From Recovery Ability

Abstract

Freezing temperature is a key environmental factor that influences plant growth and distribution. Mosses exhibit remarkable resistance to freezing stress due to their unique morphological and physiological traits. The protonema, which is the initial structure formed during the germination of a moss spore, exhibits a short life cycle and is highly sensitive to environmental changes. In this study, the protonemas of three moss species, Physcomitrium patens, Bryum argenteum, and Syntrichia caninervis, were harvested when they were 5, 10, and 15 days old. Protonemas were air dried for 0, 1, 2, and 12 h. Air-dried protonemas were kept at -80°C for 6 months to evaluate their resilience to ultra-low freezing stress. This resilience was assessed at 6, 12, and 18 days after re-culture. The three moss species exhibited varying degrees of freezing tolerance. P. patens did not recover after -80°C treatment, fully dried 10-days-old B. argenteum achieved highest recovery rate of 99.6% ± 0.2% while fully dried 5-days-old S. caninervis achieved the highest recovery rate of 98.6% ± 0.5%. The regeneration rate was influenced by both relative water content (RWC) and age. An analysis using a linear mixed-effects model indicated that the impact of RWC (effect size = 0.75) was greater than that of age (effect size = 0.35). This research provides valuable insights into the resilience of moss protonemas after exposure to -80°C, emphasizing the importance of protonema in abiotic stress research. These findings are crucial for developing methods to preserve and maintain terrestrial ecosystems in arid regions.

Keywords: age; freezing stress; moss; protonema; recovery rate; relative water content.

FIGURE 1

Diagram illustrating the growth of protonemas, ultra‐low temperature treatment, and the recovery process of three moss protonemas. (a) In vitro growth and culture under controlled conditions on Knop's medium. (b) The protonemas of three ages (5, 10, and 15 days) were air dried for 0 (control), 1, 2, and 12 h in a sterile lamina airflow at RT, then exposed to extreme low‐temperature stress. Factor 1: Different ages (5, 10, and 15 days) of protonemas. Factor 2: Air‐drying protonemas at 0, 1, 2, and 12 h, then storing them at −80°C for 6 months. (c) Thawed protonemas were grown on fresh Knop's medium to examine their ability to recover from ultra‐low temperature freezing stress.

FIGURE 2

Morphological and physiological responses of the protonemas of three moss species after different days of culture on Knop's basal medium. (a) Phenotypic response of protonemas at various time points over the 30‐day culture period. (b) Chl a, (c) Chl b, and (d) total chlorophyll content of P. patens , B. argenteum , and S. caninervis protonemas measured at 5, 10, 15, and 30 culture days. Chlorophyll was extracted by incubating the protonemas in 2 mL of 96% ethanol (25°C) for 12 h in the dark. The extracts were centrifuged at 10,000 rpm for 2 min, and the supernatants were used to quantify Chl a, Chl b, and total Chl using measurements at wavelengths of 649 nm, and 665 nm, respectively. The columns are color‐coded to represent different moss species. Data were analyzed using two‐way ANOVA at a 95% CL. Significant differences within group were determined using Fisher's LSD multiple comparison test. The data are expressed as the mean ± SD from three biological replicates. The ANOVA result represent two factors, column represent different specie and row represent age. *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 3

Changes in RWC of the protonemas of three moss species at different air‐drying time points across three ages (5, 10, and 15 days). RWC of the protonemas was measured after 0, 1, 2, and 12 h of air‐drying at room temperature (25°C) and 30%–40% relative humidity in a sterile laminar airflow cabinet to achieve fresh weight. Dry weight was determined after drying protonemas in an oven at 55°C for 48 h. (a) P. patens , (b) B. argenteum , and (c) S. caninervis . Data were analyzed using two‐way ANOVA at a 95% CL. Significant differences compared to the control (0 h) were determined using Fisher's LSD multiple comparison test. The data are expressed as the mean ± SD from three biological replicates. The ANOVA result represent two factors, column represent different protonema age and row represent air‐drying time. The ages of the protonemas are indicated by three shapes: circles, rectangles, and triangles for 5, 10, and 15 days, respectively. ***p < 0.001.

FIGURE 4

Phenotypic recovery of P. patens protonemas on Knop's medium after freezing at ultra‐low temperatures. Protonemas of different ages (5, 10, and 15 days) were air dried for 0, 1, 2, and 12 h (0 h served as the control) and then stored at −80°C in an ultra‐low temperature freezer for 6 months. After thawing, the stored P. patens protonemas were transferred onto fresh Knop's medium on Petri dishes and incubated in a growth chamber. The phenotypes of protonemas were observed at 0, 6, 12, and 18 days of recovery.

FIGURE 5

Growth recovery of B. argenteum protonemas on Knop's medium after freezing at an ultra‐low temperature. (a) In vitro growth of protonemas on Petri dishes after different air‐drying times, and of three ages. (b) Effect of RWC, age, and RWC and age on the recovery rate of protonemas using a linear mixed‐effects model. The recovery rate of protonemas was measured at 6‐day intervals starting from the day of culture. Protonemas were collected when they were 5, 10, and 15 days old and exposed to air‐drying for 0, 1, 2, and 12 h. Dried protonemas were then stored at −80°C in an ultra‐low temperature freezer for 6 months. After storage, the protonemas were transferred to fresh Knop's medium in Petri dishes and cultured in a growth chamber. Phenotypic observations were made at 0, 6, 12 and 18 days after recovery. Pink boxes highlight the highest recovery rates in response to freezing stress at −80°C. Data are presented as the mean ± SD of the estimated effect size. Statistical significance: **p < 0.01, and ***p < 0.001.

FIGURE 6

Growth recovery of S. caninervis protonemas on Knop's medium after freezing at an ultra‐low temperature. (a) In vitro growth on Petri dishes after different air‐drying times, and for three ages of protonemas. (b) Effect of RWC, age, and RWC and age on the recovery rate of protonemas using a linear mixed‐effects model. The recovery rate of protonemas was measured at 6‐day intervals starting from the day of culture. Protonemas were collected at ages of 5, 10, and 15 days and exposed to air‐drying for 0, 1, 2, and 12 h. The air‐dried protonemas were then stored at −80°C in an ultra‐low temperature freezer for 6 months. After storage, the protonemas were then transferred to fresh Knop's medium in Petri dishes and cultured in a growth chamber. Phenotypic observations were made at 0, 6, 12, and 18 days after recovery. Pink boxes highlight the highest recovery rates in response to freezing stress at −80°C. Data are presented as the mean ± SD of the estimated effect sizes. Statistical significance: **p < 0.01, and ***p < 0.001.