Pseudocrossidium replicatum (Taylor) R.H. Zander is a fully desiccation-tolerant moss that expresses an inducible molecular mechanism in response to severe abiotic stress

Abstract

The moss Pseudocrossidium replicatum is a desiccation-tolerant species that uses an inducible system to withstand severe abiotic stress in both protonemal and gametophore tissues. Desiccation tolerance (DT) is the ability of cells to recover from an air-dried state. Here, the moss Pseudocrossidium replicatum was identified as a fully desiccation-tolerant (FDT) species. Its gametophores rapidly lost more than 90% of their water content when exposed to a low-humidity atmosphere [23% relative humidity (RH)], but abscisic acid (ABA) pretreatment diminished the final water loss after equilibrium was reached. P. replicatum gametophores maintained good maximum photosystem II (PSII) efficiency (Fv/Fm) for up to two hours during slow dehydration; however, ABA pretreatment induced a faster decrease in the Fv/Fm. ABA also induced a faster recovery of the Fv/Fm after rehydration. Protein synthesis inhibitor treatment before dehydration hampered the recovery of the Fv/Fm when the gametophores were rehydrated after desiccation, suggesting the presence of an inducible protective mechanism that is activated in response to abiotic stress. This observation was also supported by accumulation of soluble sugars in gametophores exposed to ABA or NaCl. Exogenous ABA treatment delayed the germination of P. replicatum spores and induced morphological changes in protonemal cells that resembled brachycytes. Transcriptome analyses revealed the presence of an inducible molecular mechanism in P. replicatum protonemata that was activated in response to dehydration. This study is the first RNA-Seq study of the protonemal tissues of an FDT moss. Our results suggest that P. replicatum is an FDT moss equipped with an inducible molecular response that prepares this species for severe abiotic stress and that ABA plays an important role in this response.

Keywords: ABA; Bryophyte; Dehydration; Desiccation; Gametophore; Moss; Osmotic stress; Protonema; RNA-Seq; Salt stress; Transcriptome.

Fig. 1

The species P. replicatum (Taylor) R. H. Zander is widely distributed in Mexico. The pictures show the appearance of P. replicatum gametophores when hydrated (a) or dehydrated (b). Transverse hand-cut sections of leaves (c) and stems (d) are also shown. According to the registers of MEXU, P. replicatum is widely distributed across Mexico (e) but is especially enriched in the central highland regions of the country

Fig. 2

P. replicatum is an FDT moss. P. replicatum is an FDT moss that can recover its Fv/Fm to normal levels rapidly after prolonged exposure to 23% RH (black bars) or 67% RH (white bars). Totally hydrated gametophores prior to dehydration are indicated as controls for both groups of samples. The data shown are the means ± SDs from three independent experiments, with n = 5 at each indicated time

Fig. 3

Effects of ABA on dehydration kinetics and the Fv/Fm during the dehydration/rehydration process of gametophores of P. replicatum. a P. replicatum gametophores lost their water content when exposed to a low-humidity atmosphere (23% RH, solid symbols), but ABA pretreatment significantly diminished the final water loss after equilibrium was reached at this humidity level (ANOVA, p value = 0.05). The results obtained when gametophores were exposed to 67% RH are shown as open symbols. b P. replicatum gametophores maintained high Fv/Fm values for up to two hours during a slow dehydration process; however, ABA pretreatment induced a decrease in the Fv/Fm. c ABA pretreatment before dehydration resulted in a faster recovery of the Fv/Fm during the first 20 min of the rehydration process. Each point on the graph represents the average from three independent experiments, each with n = 5

Fig. 4

Inhibitor treatment reveals the participation of chloroplastic proteins required for the optimal function of PSII after dehydration. a Fv/Fm values obtained when inhibitors were applied before dehydration. b Fv/Fm responses obtained when inhibitors were applied during rehydration. Lower values of Fv/Fm were observed when CMP was applied before dehydration than when inhibitors were applied during rehydration. Each point on the graph represents the average ± SD from three independent experiments, each with n = 5. Samples showing significant differences according to ANOVA are indicated by asterisks (p value = 0.05)

Fig. 5

The gametophores of P. replicatum accumulate soluble sugars in response to ABA and abiotic stress factors. For each treatment, 100 mg of freshly collected fully hydrated gametophores were incubated for 24 h in PpNH₄ liquid medium supplemented with 400 mM sorbitol, 200 mM NaCl, or 10 µM ABA. The soluble sugars were quantified with an anthrone-sulfuric acid assay. The bars represent the averages ± SDs from three independent experiments

Fig. 6

Effects of exogenous ABA on P. replicatum spore germination and protonemal development. a Each point on the graph represents the average ± SD from three independent experiments, each with n = 120–150. Additionally, an individual protonemal filament from a monosporic line was used to analyse the influence of ABA (10 µM) on protonemal development, where b is t0 and c corresponds to 7 d of exposure