The ends of linear chromosomes of most eukaryotes consist of protein-bound DNA arrays called telomeres, which play essential roles in protecting genome integrity. Despite general evolutionary conservation in function, telomeric DNA is known to drastically vary in length and sequence between different eukaryotic lineages. Bryophytes are a group of early diverging land plants that include mosses, liverworts, and hornworts. This group of ancient land plants recently emerged as a new model for important discoveries in genomics and evolutionary biology, as well as for understanding plant adaptations to a terrestrial lifestyle. We measured telomere length in different ecotypes of model bryophyte species, including Physcomitrium patens, Marchantia polymorpha, Ceratodon purpureus, and in Sphagnum isolates. Our data indicate that all analyzed moss and liverwort genotypes have relatively short telomeres. Furthermore, all analyzed ecotypes and isolates of model mosses and liverworts display evidence of substantial natural variation in telomere length. Interestingly, telomere length also differs between male and female strains of the dioecious liverwort M. polymorpha and dioecious moss C. purpureus. Given that bryophytes are extraordinarily well adapted to different ecological niches from polar to tropical environments, our data will contribute to understanding the impact of natural telomere length variation on evolutionary adaptations in this ancient land plant lineage.

CONCLUSIONS: Characterization of Physcomitrella 3\'UTRs across different promoters yields endogenous single and double terminators for usage in molecular pharming. The production of recombinant proteins for health applications accounts for a large share of the biopharmaceutical market. While many drugs are produced in microbial and mammalian systems, plants gain more attention as expression hosts to produce eukaryotic proteins. In particular, the good manufacturing practice (GMP)-compliant moss Physcomitrella (Physcomitrium patens) has outstanding features, such as excellent genetic amenability, reproducible bioreactor cultivation, and humanized protein glycosylation patterns. In this study, we selected and characterized novel terminators for their effects on heterologous gene expression. The Physcomitrella genome contains 53,346 unique 3\'UTRs (untranslated regions) of which 7964 transcripts contain at least one intron. Over 91% of 3\'UTRs exhibit more than one polyadenylation site, indicating the prevalence of alternative polyadenylation in Physcomitrella. Out of all 3\'UTRs, 14 terminator candidates were selected and characterized via transient Dual-Luciferase assays, yielding a collection of endogenous terminators performing equally high as established heterologous terminators CaMV35S, AtHSP90, and NOS. High performing candidates were selected for testing as double terminators which impact reporter levels, dependent on terminator identity and positioning. Testing of 3\'UTRs among the different promoters NOS, CaMV35S, and PpActin5 showed an increase of more than 1000-fold between promoters PpActin5 and NOS, whereas terminators increased reporter levels by less than tenfold, demonstrating the stronger effect promoters play as compared to terminators. Among selected terminator attributes, the number of polyadenylation sites as well as polyadenylation signals were found to influence terminator performance the most. Our results improve the biotechnology platform Physcomitrella and further our understanding of how terminators influence gene expression in plants in general.

The concept of the meristem was introduced in 1858 to characterize multicellular, formative, and proliferative tissues that give rise to the entire plant body, based on observations of vascular plants. Although its original definition did not encompass bryophytes, this concept has been used and continuously refined over the past 165 years to describe the diverse apices of all land plants. Here, we re-examine this matter in light of recent evo-devo research and show that, despite displaying high anatomical diversity, land plant meristems are unified by shared genetic control. We also propose a modular view of meristem function and highlight multiple evolutionary mechanisms that are likely to have contributed to the assembly and diversification of the varied meristems during the course of plant evolution.

The phytohormone auxin, and its directional transport through tissues, plays a fundamental role in the development of higher plants. This polar auxin transport predominantly relies on PIN-FORMED (PIN) auxin exporters. Hence, PIN polarization is crucial for development, but its evolution during the rise of morphological complexity in land plants remains unclear. Here, we performed a cross-species investigation by observing the trafficking and localization of endogenous and exogenous PINs in two bryophytes, Physcomitrium patens and Marchantia polymorpha, and in the flowering plant Arabidopsis thaliana. We confirmed that the GFP fusion did not compromise the auxin export function of all examined PINs by using a radioactive auxin export assay and by observing the phenotypic changes in transgenic bryophytes. Endogenous PINs polarize to filamentous apices, while exogenous Arabidopsis PINs distribute symmetrically on the membrane in both bryophytes. In the Arabidopsis root epidermis, bryophytic PINs have no defined polarity. Pharmacological interference revealed a strong cytoskeletal dependence of bryophytic but not Arabidopsis PIN polarization. The divergence of PIN polarization and trafficking is also observed within the bryophyte clade and between tissues of individual species. These results collectively reveal the divergence of PIN trafficking and polarity mechanisms throughout land plant evolution and the co-evolution of PIN sequence-based and cell-based polarity mechanisms.

Despite decades of efforts in genome sequencing and functional characterization, some important protein families remain poorly understood. In this study, we report the classification, evolution, and functions of the largely uncharacterized AIM24 protein family in plants, including the identification of a novel subfamily. We show that two AIM24 subfamilies (AIM24-A and AIM24-B) are commonly distributed in major plant groups. These two subfamilies not only have modest sequence similarities and different gene structures but also are of independent bacterial ancestry. We performed comparative functional investigations on the two AIM24 subfamilies using three model plants: the moss Physcomitrium patens, the liverwort Marchantia polymorpha, and the flowering plant Arabidopsis thaliana. Intriguingly, despite their significant differences in sequence and gene structure, both AIM24 subfamilies are involved in ER stress tolerance and the unfolded protein response (UPR). In addition, transformation of the AIM24-A gene from P. patens into the AIM24-B null mutant of A. thaliana could at least partially rescue ER stress tolerance and the UPR. We also discuss the role of AIM24 genes in plant development and other cellular activities. This study provides a unique example of parallel evolution in molecular functions and can serve as a foundation for further investigation of the AIM24 family in plants.

Leaf senescence accompanied by yellowing and Rubisco degradation occurs prematurely in response to various stresses. However, signaling pathways between stress perception and senescence responses are not understood fully, although previous studies suggest the involvement of reactive oxygen species (ROS). While investigating the physiological functions of autophagy in Physcomitrium patens using wild-type (WT) and autophagy-deficient atg5 strains, we found that Physcomitrium colonies senesce prematurely under dark or nitrogen-deficient conditions, with atg5 senescing earlier than WT. In the present study, we measured cellular H2O2, and examined whether H2O2 mediates premature senescence in Physcomitrium colonies. Methyl viologen, an ROS generator, increased cellular H2O2 levels and caused senescence-like symptoms. H2O2 levels were also elevated to the same plateau levels in WT and atg5 under dark or nitrogen-deficient conditions. The ROS scavenger N-acetylcysteine and the ROS source inhibitor carbonyl cyanide m-chlorophenylhydrazone inhibited the increase in H2O2 levels as well as senescence. Upon transfer to a nitrogen-deficient medium, H2O2 levels increased earlier in atg5 than in WT by ~18 h, whereas atg5 yellowed earlier by >2 days. We conclude that the increased H2O2 levels under dark or nitrogen-deficient conditions mediate premature senescence in Physcomitrium but do not explain the different senescence responses of WT and atg5 cells.

CONCLUSIONS: Exogenous BAP but not 2iP disrupts actin structures and induces tip-growth retardation and cytokinesis failure in the moss Physcomitrium patens. Synthetic cytokinins have been widely used to address hormonal responses during plant development. However, exogenous cytokinins can cause a variety of cellular effects. A detailed characterization of such effects has not been well studied. Here, using Physcomitrium patens as a model, we show that the aromatic cytokinin 6-benzylaminopurine (BAP) inhibits tip growth at concentrations above 0.2 µM. At higher concentrations (0.6-1 µM), BAP can additionally block mitotic entry and induce cytokinesis defects and cell death. These effects are associated with altered actin dynamics and structures. By contrast, 2-isopentenyladenine (2iP) does not cause marked defects at various concentrations up to 10 µM, while t-zeatin (tZ) can moderately inhibit moss growth. Our results provide mechanistic insight into the inhibitory effects of BAP on cell growth and cell division and call for attention to the use of synthetic cytokinins for bioassays.

Nijmegen breakage syndrome 1 (NBS1) protein is a core member of the MRE11-RAD50-NBS1 (MRN) complex that plays a crucial role in DNA damage sensing and repair in plants. Here we report that NBS1 from moss Physcomitrium patens reduces oxidative damage by lowering the cellular ROS in addition to its known role in oxidative DNA damage recovery. Real-time transcript analysis showed up-regulation of the PpNBS1 transcript under different stress conditions. Bacterial cells showed better cell survivability upon over-expressing PpNBS1 protein as compared to untransformed cells. Likewise, overexpression of PpNBS1 in tobacco plants provides improved protection against oxidative damage and exhibited a lesser amount of ROS upon exposure to oxidative stress. Moreover, PpNBS1 contributes to the antioxidant defense mechanism by positively regulating the expression of the antioxidant genes under stress conditions in transgenic tobacco plants. PpNBS1 expressing transgenic tobacco plants resulted in lesser membrane damage, lower lipid peroxidation level, and higher chlorophyll content under stress conditions. Taken together, we conclude in addition to its known role as DNA damage sensor, PpNBS1 also plays a definite role in oxidative stress mitigation by minimizing ROS accumulation in the cell.

The PIEZO protein family was first described in animals where these mechanosensitive calcium channels perform numerous essential functions, including the perception of light touch, shear, and compressive forces. PIEZO homologs are present in most eukaryotic lineages and recently we reported that two PIEZO homologs from moss Physcomitrium patens localize to the vacuolar membrane and modulate its morphology in tip-growing caulonemal cells. Here we show that predicted structures of both PpPIEZO1 and PpPIEZO2 are very similar to that of mouse Piezo2. Furthermore, we show that both moss PIEZO genes are ubiquitously expressed in moss vegetative tissues and that they are not required for normal vacuolar pH or intracellular osmotic potential. These results suggest that moss PIEZO proteins are widely expressed mechanosensory calcium channels that serve a signaling rather than maintenance role in vacuoles.

UNASSIGNED: In Physcomitrella, whole-genome duplications affected the expression of about 3.7% of the protein-encoding genes, some of them relevant for DNA repair, resulting in a massively reduced gene-targeting frequency. Qualitative changes in gene expression after an autopolyploidization event, a pure duplication of the whole genome (WGD), might be relevant for a different regulation of molecular mechanisms between angiosperms growing in a life cycle with a dominant diploid sporophytic stage and the haploid-dominant mosses. Whereas angiosperms repair DNA double-strand breaks (DSB) preferentially via non-homologous end joining (NHEJ), in the moss Physcomitrella homologous recombination (HR) is the main DNA-DSB repair pathway. HR facilitates the precise integration of foreign DNA into the genome via gene targeting (GT). Here, we studied the influence of ploidy on gene expression patterns and GT efficiency in Physcomitrella using haploid plants and autodiploid plants, generated via an artificial WGD. Single cells (protoplasts) were transfected with a GT construct and material from different time-points after transfection was analysed by microarrays and SuperSAGE sequencing. In the SuperSAGE data, we detected 3.7% of the Physcomitrella genes as differentially expressed in response to the WGD event. Among the differentially expressed genes involved in DNA-DSB repair was an upregulated gene encoding the X-ray repair cross-complementing protein 4 (XRCC4), a key player in NHEJ. Analysing the GT efficiency, we observed that autodiploid plants were significantly GT suppressed (p < 0.001) attaining only one third of the expected GT rates. Hence, an alteration of global transcript patterns, including genes related to DNA repair, in autodiploid Physcomitrella plants correlated with a drastic suppression of HR.