Drought responses and population differentiation of Calohypnum plumiforme inferred from comparative transcriptome analysis.

Bryophytes, known as poikilohydric plants, possess vegetative desiccation-tolerant (DT) ability to withstand water deficit stress. Consequently, they offer valuable genetic resources for enhancing resistance to water scarcity stress. In this research, we examined the physiological, phytohormonal, and transcriptomic changes in DT mosses Calohypnum plumiforme from two populations, with and without desiccation treatment. Comparative analysis revealed population differentiation at physiological, gene sequence, and expression levels. Under desiccation stress, the activities of superoxide dismutase (SOD) and peroxidase (POD) showed significant increases, along with elevation of soluble sugars and proteins, consistent with the transcriptome changes. Notable activation of the bypass pathway of JA biosynthesis suggested their roles in compensating for JA accumulation. Furthermore, our analysis revealed significant correlations among phytohormones and DEGs in their respective signaling pathway, indicating potential complex interplays of hormones in C plumiforme. Protein phosphatase 2C (PP2C) in the abscisic acid signaling pathway emerged as the pivotal hub in the phytohormone crosstalk regulation network. Overall, this study was one of the first comprehensive transcriptome analyses of moss C. plumiforme under slow desiccation rates, expanding our knowledge of bryophyte transcriptomes and shedding light on the gene regulatory network involved in response to desiccation, as well as the evolutionary processes of local adaptation across moss populations.

Evidence of stress imprinting with population-level differences in two moss species.

Plants are often repeatedly exposed to stresses during their lives and have a mechanism called stress imprinting that provides "memories" of stresses they experience and increases their ability to cope with later stresses. To test hypotheses that primed bryophytes can preserve their stress imprinting after 6 days of recovery and induce higher levels of osmolytes and ROS-scavenging activities upon later stress exposure, and there exist population-level differentiation in their desiccation defenses, we transplanted samples of two populations of each of two moss species, Hypnum plumaeforme and Pogonatum cirratum, in a nature reserve in southern China. After 16 months of acclimation, sets of each population were subjected to control, one-time desiccation stress, duplicated desiccation stress and cross-stress (low temperature stress followed by desiccation stress) treatments. Levels of oxidant enzymes, osmolytes, and phytohormones in the samples were then determined. The desiccation stress generally led to increases in activities or contents of superoxide dismutase, guaiacol peroxidase, catalase, proline, soluble sugars, soluble proteins, and stress hormones including abscisic acid (ABA), jasmonates (JA), and salicylic acid (SA), with differences between both species and populations. After a 6-day recovery period, contents of phytohormones (including ABA, JA, SA, and cytokinins) in stressed H. plumaeforme had substantially fallen toward control levels. The duplicated and cross-stress treatments generally led to further accumulation of proline, soluble sugars, and soluble proteins, with further increases in activities of antioxidant enzymes in some cases. Furthermore, significant differences between allochthonous and native populations were found in contents of malondialdehyde and osmolytes, as well as antioxidant enzyme activities. Our results confirm the hypotheses and highlight the importance of osmolytes in mosses' stress responses.

Nitrogen Addition Exacerbates the Negative Effects of Low Temperature Stress on Carbon and Nitrogen Metabolism in Moss.

Global environmental changes are leading to an increase in localized abnormally low temperatures and increasing nitrogen (N) deposition is a phenomenon recognized worldwide. Both low temperature stress (LTS) and excess N induce oxidative stress in plants, and excess N also reduces their resistance to LTS. Mosses are primitive plants that are generally more sensitive to alterations in environmental factors than vascular species. To study the combined effects of N deposition and LTS on carbon (C) and N metabolism in moss, two moss species, Pogonatum cirratum subsp. fuscatum, and Hypnum plumaeforme, exposed to various concentrations of nitrate (KNO3) or ammonium (NH4Cl), were treated with or without LTS. C/N metabolism indices were then monitored, both immediately after the stress and after a short recovery period (10 days). LTS decreased the photosystem II (PSII) performance index and inhibited non-cyclic photophosphorylation, ribulose-1,5-bisphosphate carboxylase, and glutamine synthetase activities, indicating damage to PSII and reductions in C/N assimilation in these mosses. LTS did not affect cyclic photophosphorylation, sucrose synthase, sucrose-phosphate synthase, and NADP-isocitrate dehydrogenase activities, suggesting a certain level of energy and C skeleton generation were maintained in the mosses to combat LTS; however, LTS inhibited the activity of glycolate oxidase. As predicted, N supply increased the sensitivity of the mosses to LTS, resulting in greater damage to PSII and a sharper decrease in C/N assimilation. After the recovery period, the performance of PSII and C/N metabolism, which were inhibited by LTS increased significantly, and were generally higher than those of control samples not exposed to LTS, suggesting overcompensation effects; however, N application reduced the extent of compensation effects. Both C and N metabolism exhibited stronger compensation effects in H. plumaeforme than in P. cirratum subsp. fuscatum. The difference was especially pronounced after addition of N, indicating that H. plumaeforme may be more resilient to temperature and N variation, which could explain its wider distribution in the natural environment.

Physiological responses of two moss species to the combined stress of water deficit and elevated N deposition (II): Carbon and nitrogen metabolism.

Nitrogen (N) deposition levels and frequencies of extreme drought events are increasing globally. In efforts to improve understanding of plants' responses to associated stresses, we have investigated responses of mosses to drought under elevated nitrogen conditions. More specifically, we exposed Pogonatum cirratum subsp. fuscatum and Hypnum plumaeforme to various nitrate (KNO 3) or ammonium (NH 4Cl) treatments, with and without water deficit stress and monitored indices related to carbon (C) and N metabolism both immediately after the stress and after a short recovery period. The results show that N application stimulated both C and N assimilation activities, including ribulose-1,5-bisphosphate carboxylase, glutamine synthetase/glutamate synthase (GS/GOGAT), and glutamate dehydrogenase (GDH) activities, while water deficit inhibited C and N assimilation. The mosses could resist stress caused by excess N and water deficit by increasing their photorespiration activity and proline (Pro) contents. However, N supply increased their sensitivity to water stress, causing sharper reductions in C and N assimilation rates, and further increases in photorespiration and Pro contents, indicating more serious oxidative or osmotic stress in the mosses. In addition, there were interspecific differences in N assimilation pathways, as the GS/GOGAT and GDH pathways were the preferentially used ammonium assimilation pathways in P. cirratum and H. plumaeforme when stressed, respectively. After rehydration, both mosses exhibited overcompensation effects for most C and N assimilation activities, but when supplied with N, the activities were generally restored to previous levels (or less), indicating that N supply reduced their ability to recover from water deficit stress. In conclusion, mosses can tolerate a certain degree of water deficit stress and possess some resilience to environmental fluctuations, but elevated N deposition reduces their tolerance and ability to recover.