Search

ent-Kaurene is a biosynthetic intermediate diterpene of phytohormone gibberellins, and is biosynthesized from geranylgeranyl diphosphate via ent-copalyl diphosphate (ent-CDP). The successive cyclization is catalyzed by two distinct diterpene synthases, ent-CDP synthase (ent-CPS) and ent-kaurene synthase (KS). Homologs of these diterpene synthase genes have been reported to be involved in the biosynthesis of specialized-metabolic diterpenoids for defense in several plant species, including rice (Oryza sativa). These diterpene synthases consist of three domains, αβγ domains. Active sites of ent-CPS exist at the interface of β and γ domain, while those of KS are located within the α domain. We herein carried out domain-deletion experiments using several KSs and KS like enzymes (KSLs) to obtain insights into the roles of domains other than active-site domains. As previously reported in taxadiene synthase, deletion of γ or βγ domains drastically decreased activities of specialized-metabolic OsKSL5, OsKSL8, OsKSL7 and OsKSL10 in O. sativa. However, unexpectedly, only α domains of several gibberellin-biosynthetic KSs, including OsKS1 in O. sativa, AtKS in Arabidopsis thaliana, TaKS in wheat (Triticum aestivum) and BdKS1 in Brachypodium distachyon, retained their original functions. Additionally, the specialized-metabolic OsKSL4, which is closely related to OsKS1, also functioned without its βγ domains. Domain-swapping experiments showed that replacing βγ domains in OsKSL7 with those from other KS/KSLs retained the OsKSL7 activity. Moreover, deletion of βγ domains of bifunctional PpCPS/KS in moss (Physcomitrella patens) drastically impaired its KS-related activity. Thus, we demonstrate that monofunctional gibberellinbiosynthetic KSs are the unique diterpene synthases that retain their functions without βγ domains. [ABSTRACT FROM AUTHOR]

Zinc-finger homeodomain transcription factors (ZF-HD TFs) are relatively a small gene family in Arabidopsis involved in plant development and stress response. However, the biological functions of ZF-HD TFs remain largely undiscovered. Here, we aimed to elucidate the evolutionary history and functional role of ZF-HD TFs in other species, by performing phylogenic analysis and domain and motif identification studies in Arabidopsis, sorghum (Sorghum bicolor), and moss (Physcomitrella patens). Forty-two ZF-HD TF proteins were classified into two distinct subfamilies based on the conserved ZF Cys/His-rich dimerization and homeodomain (HD) domains. The phylogenetic tree of proteins was further divided into five groups based on the similarity of sequences, and three distinct motifs were defined in the amino acid sequences. Genetic analysis revealed that the moss PpZF-HD1, Pp3c1_15290, gene partially rescued the amiR zf-HD-79 mutant lines at phenotypic and molecular levels. Subcellular localization studies revealed that moss PpZF-HD1 was localized in the cytosol and nuclei. Phylogenetic analysis and genetic complementation revealed that ZF-HD TFs play functional roles in regulating plant architecture, which is conserved in Arabidopsis, sorghum, and moss. Although our study is only a preliminary exploration into ZF-HD TFs, it provides a novel perspective that will help future researchers better understand the biological role of ZF-HD proteins in plants. [ABSTRACT FROM AUTHOR]

SUMMARY: Cells save their energy during nitrogen starvation by selective autophagy of ribosomes and degradation of RNA to ribonucleotides and nucleosides. Nucleosides are hydrolyzed by nucleoside N‐ribohydrolases (nucleosidases, NRHs). Subclass I of NRHs preferentially hydrolyzes the purine ribosides while subclass II is more active towards uridine and xanthosine. Here, we performed a crystallographic and kinetic study to shed light on nucleoside preferences among plant NRHs followed by in vivo metabolomic and phenotyping analyses to reveal the consequences of enhanced nucleoside breakdown. We report the crystal structure of Zea mays NRH2b (subclass II) and NRH3 (subclass I) in complexes with the substrate analog forodesine. Purine and pyrimidine catabolism are inseparable because nucleobase binding in the active site of ZmNRH is mediated via a water network and is thus unspecific. Dexamethasone‐inducible ZmNRH overexpressor lines of Arabidopsis thaliana, as well as double nrh knockout lines of moss Physcomitrium patents, reveal a fine control of adenosine in contrast to other ribosides. ZmNRH overexpressor lines display an accelerated early vegetative phase including faster root and rosette growth upon nitrogen starvation or osmotic stress. Moreover, the lines enter the bolting and flowering phase much earlier. We observe changes in the pathways related to nitrogen‐containing compounds such as β‐alanine and several polyamines, which allow plants to reprogram their metabolism to escape stress. Taken together, crop plant breeding targeting enhanced NRH‐mediated nitrogen recycling could therefore be a strategy to enhance plant growth tolerance and productivity under adverse growth conditions. [ABSTRACT FROM AUTHOR]

The moss Physcomitrium patens (P. patens), formerly known as Physcomitrella patens, has ascended to prominence as a pivotal model organism in plant biology. Its simplicity in structure and life cycle, coupled with genetic amenability, has rendered it indispensable in unraveling the complexities of land plant evolution and responses to environmental stimuli. As an evolutionary bridge between algae and vascular plants, P. patens offers a unique perspective on early terrestrial adaptation. This research involved the biotechnological cultivation of P. patens, followed by a deep phytochemical investigation of two extracts covering a large polarity range together using an NMR-based dereplication approach combined with GC/MS analyses. Subsequently, a multidisciplinary approach combining bioinformatics, in-silico techniques, and traditional methods was adopted to uncover intriguing molecules such as the diterpene ceruchinol and its potential receptor interactions for future cosmetic applications. The kaurene diterpene ceruchinol, representing up to 50% of the supercritical CO2 extract and also identified in the hydroalcoholic extract, was selected for the molecular docking study, which highlighted several biological targets as CAR, AKR1D1, and 17β-HSD1 for potential cosmetic use. These findings offer valuable insights for novel uses of this plant biomass in the future. [ABSTRACT FROM AUTHOR]

Crosstalk between auxin and cytokinin contributes to widespread developmental processes, including root and shoot meristem maintenance, phyllotaxy, and vascular patterning. However, our understanding of crosstalk between these hormones is limited primarily to angiosperms. The moss Physcomitrium patens (formerly Physcomitrella patens) is a powerful system for studying plant hormone function. Auxin and cytokinin play similar roles in regulating moss gametophore (shoot) architecture, to those in flowering plant shoots. However, auxin–cytokinin crosstalk is poorly understood in moss. Here we find that the ratio of auxin to cytokinin is an important determinant of development in P. patens , especially during leaf development and branch stem cell initiation. Addition of high levels of auxin to P. patens gametophores blocks leaf outgrowth. However, simultaneous addition of high levels of both auxin and cytokinin partially restores leaf outgrowth, suggesting that the ratio of these hormones is the predominant factor. Likewise, during branch initiation and outgrowth, chemical inhibition of auxin synthesis phenocopies cytokinin application. Finally, cytokinin-insensitive mutants resemble plants with altered auxin signaling and are hypersensitive to auxin. In summary, our results suggest that the ratio between auxin and cytokinin signaling is the basis for developmental decisions in the moss gametophore. [ABSTRACT FROM AUTHOR]

MutS homolog 1 (MSH1) is involved in the recombining and repairing of organelle genomes and is essential for maintaining their stability. Previous studies indicated that the length of the gene varied greatly among species and detected species-specific partial gene duplications in Physcomitrella patens. However, there are critical gaps in the understanding of the gene size expansion, and the extent of the partial gene duplication of MSH1 remains unclear. Here, we screened MSH1 genes in 85 selected species with genome sequences representing the main clades of green plants (Viridiplantae). We identified the MSH1 gene in all lineages of green plants, except for nine incomplete species, for bioinformatics analysis. The gene is a singleton gene in most of the selected species with conserved amino acids and protein domains. Gene length varies greatly among the species, ranging from 3234 bp in Ostreococcus tauri to 805,861 bp in Cycas panzhihuaensis. The expansion of MSH1 repeatedly occurred in multiple clades, especially in Gymnosperms, Orchidaceae, and Chloranthus spicatus. MSH1 has exceptionally long introns in certain species due to the gene length expansion, and the longest intron even reaches 101,025 bp. And the gene length is positively correlated with the proportion of the transposable elements (TEs) in the introns. In addition, gene structure analysis indicated that the MSH1 of green plants had undergone parallel intron gains and losses in all major lineages. However, the intron number of seed plants (gymnosperm and angiosperm) is relatively stable. All the selected gymnosperms contain 22 introns except for Gnetum montanum and Welwitschia mirabilis, while all the selected angiosperm species preserve 21 introns except for the ANA grade. Notably, the coding region of MSH1 in algae presents an exceptionally high GC content (47.7% to 75.5%). Moreover, over one-third of the selected species contain species-specific partial gene duplications of MSH1, except for the conserved mosses-specific partial gene duplication. Additionally, we found conserved alternatively spliced MSH1 transcripts in five species. The study of MSH1 sheds light on the evolution of the long genes of green plants. [ABSTRACT FROM AUTHOR]

There exists an obvious gap in our knowledge of the nuclear DNA amount of bryophytes, not only in terms of the low number of species represented, but also in systematic and geographic representation. In order to increase our knowledge of nuclear DNA amounts and variation patterns in bryophytes, and their potential phylogenetic significances and influences on phenotypes, we used flow cytometry to determine the DNA 1C values of 209 bryophyte accessions, which belong to 145 mosses and 18 liverworts collected from China, by using Physcomitrella patens as a standard. We quantified the differences in DNA 1C values among different orders and families and constructed a phylogenetic tree of 112 mosses with four gene sequences (nad5, rbcL, trnL-F, and 18S-ITS1-5.8S-ITS2-26S). DNA 1C values were mapped onto the phylogenetic tree to test a potential phylogenetic signal. We also evaluated the correlations of the DNA 1C value with the sizes of individuals, leaves, cells, and spores by using a phylogenetically controlled analysis. New estimates of nuclear DNA amounts were reported for 145 species. The DNA 1C values of 209 bryophyte accessions ranged from 0.422 pg to 0.860 pg, with an average value of 0.561 pg, and a 2.04-fold variation covered the extremes of all the accessions. Although the values are not significantly different (p = 0.355) between mosses (0.528 pg) and liverworts (0.542 pg), there are variations to varying extents between some families and orders. The DNA 1C value size exerts a positive effect on the sizes of plants, leaves, and cells, but a negative effect on spore size. A weak phylogenetic signal is detected across most moss species. Phylogenetic signals are comparatively strong for some lineages. Our findings show that bryophytes have very small and highly constrained nuclear DNA amounts. There are nucleotype effects of nuclear DNA amounts for bryophytes at the individual, organ, and cell levels. We speculate that smaller nuclear DNA amounts are advantageous for bryophytes in dry environments. Significant differences in the DNA 1C values among some moss families and orders, as well as phylogenetic signals for some lineages, imply that nuclear DNA amount evolution in mosses seems to be unidirectional. [ABSTRACT FROM AUTHOR]

Syntrichia caninervis can survive under 80–90% protoplasmic water losses, and it is a model plant in desiccation tolerance research. A previous study has revealed that S. caninervis would accumulate ABA under dehydration stress, while the ABA biosynthesis genes in S. caninervis are still unknown. This study identified one ScABA1, two ScABA4s, five ScNCEDs, twenty-nine ScABA2s, one ScABA3, and four ScAAOs genes, indicating that the ABA biosynthesis genes were complete in S. caninervis. Gene location analysis showed that the ABA biosynthesis genes were evenly distributed in chromosomes but were not allocated to sex chromosomes. Collinear analysis revealed that ScABA1, ScNCED, and ScABA2 had homologous genes in Physcomitrella patens. RT-qPCR detection found that all of the ABA biosynthesis genes responded to abiotic stress; it further indicated that ABA plays an important role in S. caninervis. Moreover, the ABA biosynthesis genes in 19 representative plants were compared to study their phylogenetic and conserved motifs; the results suggested that the ABA biosynthesis genes were closely associated with plant taxa, but these genes had the same conserved domain in each plant. In contrast, there is a huge variation in the exon number between different plant taxa; it revealed that ABA biosynthesis gene structures are closely related to plant taxa. Above all, this study provides strong evidence demonstrating that ABA biosynthesis genes were conserved in the plant kingdom and deepens our understanding of the evolution of the phytohormone ABA. [ABSTRACT FROM AUTHOR]

The vast diversity of traits exhibited by horticultural crops largely depends upon variation in gene expression regulation. The uppermost layer of gene expression regulation is chromatin compaction. In plants, the LIKE HETEROCHROMATIN PROTEIN 1 (LHP1) is a member of the Polycomb Repressive Complex 1 (PRC1) that controls the spreading of the H3K27me3 mark throughout the genome to regulate gene expression. Much of the epigenetic control exerted by LHP1 has been deeply explored on the model species Arabidopsis thaliana. Recent advances in melon, tomato, and soybean highlight the relevance of LHP1 in controlling the development and physiology of a plethora of traits in crops. However, whether LHP1 exerts its diverse roles through similar mechanisms and through modulating the same target genes has been overlooked. In this review, we gather a wealth of knowledge about the LHP1 mode of action, which involves a tight connection with histone marks and long noncoding RNAs to modulate gene expression. Strikingly, we found that LHP1 may be linked to H3K27me3 regulation across the plant lineage, yet, through epigenetic regulation of a distinct set of target genes. This is supported by subtle differences in subcellular LHP1 localization between species found here. In addition, we summarize the variety of developmental outputs modulated by LHP1 across land plants pinpointing its importance for plant breeding. Hence, LHP1 has probably been co-opted in different lineages to modulate diverse traits contributing to crop diversification. [ABSTRACT FROM AUTHOR]

Plant oxylipins are a class of lipid-derived signaling molecules being involved in the regulation of various biotic and abiotic stress responses. A major class of oxylipins are the circular derivatives to which 12-oxo-phytodienoic acid (OPDA) and its metabolite jasmonic acid (JA) belong. While OPDA and its shorter chain homologue dinor-OPDA (dnOPDA) seem to be ubiquitously found in land plants ranging from bryophytes to angiosperms, the occurrence of JA and its derivatives is still under discussion. The bryophyte Physcomitrium patens has received increased scientific interest as a nonvascular plant model organism over the last decade. Therefore, we followed the metabolism upon wounding by metabolite fingerprinting with the aim to identify jasmonates as well as novel oxylipins in P. patens. A non-targeted metabolomics approach was used to reconstruct the metabolic pathways for the synthesis of oxylipins, derived from roughanic, linoleic, a-linolenic, and arachidonic acid in wild type, the oxylipin-deficient mutants of Ppaos1 and Ppaos2, the mutants of Ppdes being deficient in all fatty acids harboring a D6- double bond and the C20-fatty acid-deficient mutants of Ppelo. Beside of OPDA, iso-OPDA, dnOPDA, and iso-dnOPDA, three additional C18- compounds and a metabolite being isobaric to JA were identified to accumulate after wounding. These findings can now serve as foundation for future research in determining, which compound(s) will serve as native ligand(s) for the oxylipin-receptor COI1 in P. patens. [ABSTRACT FROM AUTHOR]

Plant cells form microtubule arrays, called 'cortical microtubules', beneath the plasma membrane which are critical for cell‐wall organization and directional cell growth. Cortical microtubules are nucleated independently of centrosomes. Spiral2 is a land‐plant‐specific microtubule minus‐end‐targeting protein that stabilizes the minus ends by inhibiting depolymerization of the filament. Spiral2 possesses an N‐terminal microtubule‐binding domain and a conserved C‐terminal domain whose function is unknown. In this study, the crystal structure of the conserved C‐terminal domain of Spiral2 was determined using the single‐wavelength anomalous dispersion method. Refinement of the model to a resolution of 2.2 Å revealed a helix–turn–helix fold with seven α‐helices. The protein crystallized as a dimer, but SEC‐MALS analysis showed the protein to be monomeric. A structural homology search revealed that the protein has similarity to the C‐terminal domain of the katanin regulatory subunit p80. The structure presented here suggests that the C‐terminal domain of Spiral2 represents a new class of microtubule dynamics modulator across the kingdom. [ABSTRACT FROM AUTHOR]

Genome-wide identification of the EIN3 / EIL gene family in Ginkgo biloba and functional study of a GbEIL in the ethylene signaling pathway. [Display omitted] • Five GbEIL s are differentially expressed with high, low, and moderate levels. • A GbEIL (Gb_03292) modulates the expression of a GbERF by binding to its promoter. • Gb_03292 rescues the defect of triple response to ethylene in ein3eil1 Arabidopsis. • Gb_03292 has the evolutionary conservation in ethylene signaling. ETHYLENE-INSENSITIVE3 (EIN3) or EIN3-Like (EIL) proteins, play critical roles in integrating ethylene signaling and physiological regulation in plants by modulating the expression of various downstream genes, such as ethylene-response factors (ERFs). However, little is known about the characteristics of EIN3/EILs in the gymnosperm Ginkgo biloba. In the present study, a genome-wide comparative analysis of Ginkgo EIN3 / EIL gene family was performed with those from an array of species, including bryophytes (Physcomitrella patens), gymnosperms (Cycas panzhihuaensis), and angiosperms (Arabidopsis thaliana , Gossypium raimondii , Gossypium hirsutum , Oryza sativa , and Brachypodium distachyon). Within the constructed phylogenetic tree for the 53 EIN3/EILs identified, 5 GbEILs from G. biloba , 2 PpEILs from P. patens , and 3 CpEILs from C. panzhihuaensis were assigned to one cluster, suggesting that their derivation occurred after the split of their ancestors and angiosperms. Although considerable divergence accumulated in amino acid sequences along with the evolutionary process, the specific EIN3_DNA-binding domains were evolutionarily conserved among the 53 EIN3/EILs. Collinearity analysis indicated that whole-genome or segmental duplication and subsequent purifying selection might have prompted the generation and evolution of EIN3 / EIL multigene families. Based on the expression patterns of five GbEIL s at the four developmental stages of Ginkgo ovules, one GbEIL gene (Gb_03292) was further investigated for its role in mediating ethylene signaling. The functional activity of Gb_03292 was closely related to ethylene signaling, as it complemented the triple response via ectopic expression in ein3eil1 double mutant Arabidopsis. Additionally, GbEIL likely modulates the expression of a Ginkgo ERF (Gb_15517) by directly binding to its promoter. These results demonstrated that the GbEIL gene could have participated in mediating ethylene signal transduction during ovule development in G. biloba. The present study also provides insights into the conservation of ethylene signaling across the gymnosperm G. biloba and angiosperm species. [ABSTRACT FROM AUTHOR]

Plant oxylipins are a class of lipid-derived signaling molecules being involved in the regulation of various biotic and abiotic stress responses. A major class of oxylipins are the circular derivatives to which 12-oxo-phytodienoic acid (OPDA) and its metabolite jasmonic acid (JA) belong. While OPDA and its shorter chain homologue dinor-OPDA (dnOPDA) seem to be ubiquitously found in land plants ranging from bryophytes to angiosperms, the occurrence of JA and its derivatives is still under discussion. The bryophyte Physcomitrium patens has received increased scientific interest as a non-vascular plant model organism over the last decade. Therefore, we followed the metabolism upon wounding by metabolite fingerprinting with the aim to identify jasmonates as well as novel oxylipins in P. patens. A non-targeted metabolomics approach was used to reconstruct the metabolic pathways for the synthesis of oxylipins, derived from roughanic, linoleic, α-linolenic, and arachidonic acid in wild type, the oxylipin-deficient mutants of Ppaos1 and Ppaos2, the mutants of Ppdes being deficient in all fatty acids harboring a Δ6-double bond and the C20-fatty acid-deficient mutants of Ppelo. Beside of OPDA, iso-OPDA, dnOPDA, and iso-dnOPDA, three additional C18-compounds and a metabolite being isobaric to JA were identified to accumulate after wounding. These findings can now serve as foundation for future research in determining, which compound(s) will serve as native ligand(s) for the oxylipin-receptor COI1 in P. patens.

Plant phospholipase D (PLD) exerts important roles in various biological processes, such as intracellular signaling and morphological development. Our knowledge about early land plant PLDs is still underdeveloped. In this study, we identified 84 PLD genes in six mosses, i.e., Physcomitrella patens, Ceratodon purpureus, Fontinalis antipyretica, Pleurozium schreberi, Sphagnum magellanicum, and Sphagnum fallax. These PLDs were classified into four clades (I-IV). We showed that PLD underwent rapid expansion in mosses. A total of six conserved domains and two core HKD motifs were detected. Structure analysis uncovered that the moss PLDs from within a clade generally exhibited similar exon-intron organization. Cis-elements prediction and expression analyses indicated that P. patens PLDs had key roles in stress responsiveness and plant development. Particularly, about half of the P. patens PLDs (e.g., PpPLD1, PpPLD2, and PpPLD5) were differentially expressed under biotic and abiotic stresses. We also determined the expression pattern of P. patens PLD genes in various tissues and at different stages of development. Although the moss, clubmoss, liverwort, and fern PLDs evolved largely under functional constraints, we found episodic positive selection in the moss PLDs, e.g., C. purpureus PLD2 and P. patens PLD11. We infer that the evolutionary force acting on the PLDs may have facilitated moss colonization of land. Our work provides valuable insights into the diversification of moss PLD genes, and can be used for future studies of their functions. [ABSTRACT FROM AUTHOR]

A study published in the journal Plant Cell explores the role of SUPPRESSOR OF MAX2 (SMAX)1-LIKE (SMXL) proteins in regulating growth in the moss Physcomitrium patens. The researchers found that SMXL proteins act as negative growth regulators in the moss, functioning downstream of MAX2 in the KL signaling pathway. They also discovered that SMXL proteins do not act as repressors in the strigolactone (SL) pathway. The study provides insights into the molecular mechanisms of moss growth regulation and suggests a possible interplay between SL and KL signaling pathways in moss development. [Extracted from the article]

Plant cell wall proteins (CWPs) play critical roles during plant development and in response to stresses. Proteomics has revealed their great diversity. With nearly 1000 identified CWPs, the Arabidopsis thaliana cell wall proteome is the best described to date and it covers the main plant organs and cell suspension cultures. Other monocot and dicot plants have been studied as well as bryophytes, such as Physcomitrella patens and Marchantia polymorpha. Although these proteomes were obtained using various flowcharts, they can be searched for the presence of members of a given protein family. Thereby, a core cell wall proteome which does not pretend to be exhaustive, yet could be defined. It comprises: (i) glycoside hydrolases and pectin methyl esterases, (ii) class III peroxidases, (iii) Asp, Ser and Cys proteases, (iv) non-specific lipid transfer proteins, (v) fasciclin arabinogalactan proteins, (vi) purple acid phosphatases and (vii) thaumatins. All the conserved CWP families could represent a set of house-keeping CWPs critical for either the maintenance of the basic cell wall functions, allowing immediate response to environmental stresses or both. Besides, the presence of non-canonical proteins devoid of a predicted signal peptide in cell wall proteomes is discussed in relation to the possible existence of alternative secretion pathways. [ABSTRACT FROM AUTHOR]

The purpose of this study was to explore the structural and expression characteristics of LEA2 genes from Brachymenium exile. BeLEA2 gene was firstly isolated and analyzed by polymerase chain reaction(PCR). The results were as follows:(1)Gene structure analysis showed that BeLEA2 gene contained 2 exons and 1 intron and contained an open reading frame(ORF)of 456 bp encoding a protein of 151 amino acids, and its molecular mass was predicted to be 16 515.96 Da.(2)The phylogenetic analysis of LEA2 with other LEA2 in different plants revealed that BeLEA2 from B. exile and LEA2 from Physcomitrella patens belonged to the same branch of evolutionary distance.(3)The promoter sequence of the BeLEA2 gene of 1 072 bp was isolated from Brachymenium exile by high-efficiency hermal asymmetric interlaced polymerase chain reaction (HiTail-PCR) and analyzed by PlantCARE, the results showed that it had TATA-box, CAAT-box, ABRE, MYB, MYC, MYB binding site and other cis-acting elements.(4)Quantitative real-time PCR analysis indicated that BeLEA2 expressed in different stages and tissues of B. exile, and BeLEA2 responded to dehydration stress. These results lay a foundation for further study on the function of LEA2 gene in bryophytes. [ABSTRACT FROM AUTHOR]

As a transcription factor first found in plants, WRKY plays an important role in plant growth and development. However, it has not been studied in Physcomitrella patens. By using the WRKY gene family data(accession number is PF03106) in Pfam database, this paper studied the basic information of WRKY in P. patens, which included physicochemical properties, protein secondary structure prediction, chromosome localization, exon and intron distribution and phylogenetic relationship. The results were as follows:(1)The WRKY gene family in P. patens consisted of 38 members which were divided into two major categories ─Ⅰ and Ⅱ, and the conserved domains of some WRKY genes had mutated.(2)The amino acid length of WRKY protein was 216-775 aa and the relative molecular mass was 24.5-82.8 kDa; Subcellular location showed that the proteins of WRKY gene family were distributed in the nucleus.(3)The secondary structure of WRKY protein was composed of four constituent elements: α-helix, extended strand, β-turn, and random coil; Except for PpWRKY11(α-helix dominated), the proportion of random coil was up to 70%.(4)The phylogenetic relationship with Arabidopsis showed that the number of the WRKY family members and the way of their revolution had changed in the process of plants' evolution, the number of exons of WRKY gene family members were 3-7.(5)The members of WRKY gene were randomly dispersed on 21 chromosomes without forming a gene cluster. This study analyzed the basic structure and properties of the WRKY gene family, and it was found that the WRKY gene family of Physcomitrella patens has evolutionary diversity and unique variability in conserved domains, laying the foundation for subsequent research. [ABSTRACT FROM AUTHOR]

Plant viruses are important pathogens able to overcome plant defense mechanisms using their viral suppressors of RNA silencing (VSR). Small RNA pathways of bryophytes and vascular plants have significant similarities, but little is known about how viruses interact with mosses. This study elucidated the responses of Physcomitrella patens to two different VSRs. We transformed P. patens plants to express VSR P19 from tomato bushy stunt virus and VSR 2b from cucumber mosaic virus, respectively. RNA sequencing and quantitative PCR were used to detect the effects of VSRs on gene expression. Small RNA (sRNA) sequencing was used to estimate the influences of VSRs on the sRNA pool of P. patens. Expression of either VSR‐encoding gene caused developmental disorders in P. patens. The transcripts of four different transcription factors (AP2/erf, EREB‐11 and two MYBs) accumulated in the P19 lines. sRNA sequencing revealed that VSR P19 significantly changed the microRNA pool in P. patens. Our results suggest that VSR P19 is functional in P. patens and affects the abundance of specific microRNAs interfering with gene expression. The results open new opportunities for using Physcomitrella as an alternative system to study plant–virus interactions. Expression of VSRs caused developmental disorders in P. patens. P. patens expressing P19 showed accumulation of four transcription factors and significant changes in the microRNA pool indicating that VSR P19 affects the abundance of specific microRNAs in P. patens. [ABSTRACT FROM AUTHOR]

Mosses (Bryophyta) are a key group occupying an important phylogenetic position in land plant (embryophyte) evolution. The class Bryopsida represents the most diversified lineage, containing more than 95% of modern mosses, whereas other classes are species‐poor. Two branches with large numbers of gene duplications were elucidated by phylogenomic analyses, one in the ancestry of all mosses and another before the separation of the Bryopsida, Polytrichopsida, and Tetraphidopsida. The analysis of the phylogenetic progression of duplicated paralogs retained on genomic syntenic regions in the Physcomitrella patens genome confirmed that the whole‐genome duplication events WGD1 and WGD2 were re‐recognized as the ψ event and the Funarioideae duplication event, respectively. The ψ polyploidy event was tightly associated with the early diversification of Bryopsida, in the ancestor of Bryidae, Dicranidae, Timmiidae, and Funariidae. Together, four branches with large numbers of gene duplications were unveiled in the evolutionary past of P. patens. Gene retention patterns following the four large‐scale duplications in different moss lineages were analyzed and discussed. Recurrent significant retention of stress‐related genes may have contributed to their adaption to distinct ecological environments and the evolutionary success of this early‐diverging land plant lineage. [ABSTRACT FROM AUTHOR]

Photosynthetic organisms have evolved photoprotective mechanisms to acclimate to light intensity fluctuations in their natural growth environments. Photosystem (PS) II subunit S (PsbS) and light-harvesting complex (LHC) stress-related proteins (LhcSR) are essential for triggering photoprotection in vascular plants and green algae, respectively. The activity of both proteins is strongly enhanced in the moss Physcomitrella patens under high-light conditions. However, their role in regulating photosynthesis acclimation in P. patens under fluctuating light (FL) conditions is still unknown. Here, we compare the responses of wild-type (WT) P. patens and mutants lacking PsbS (psbs KO) or LhcSR1 and 2 (lhcsr KO) to FL conditions in which the low-light phases were periodically interrupted with high-light pulses. lhcsr KO mutant showed a strong reduction in growth with respect to WT and psbs KO under FL conditions. The lack of LhcSR not only decreased the level of non-photochemical quenching, resulting in an over-reduced plastoquinone pool, but also significantly increased the PSI acceptor limitation values with respect to WT and psbs KO under FL conditions. Moreover, in lhcsr KO mutant, the abundance of PSI core and PSI–LHCI complex decreased greatly under FL conditions compared with the WT and psbs KO. We proposed that LhcSR in P. patens play a crucial role in moss acclimation to dynamic light changes. [ABSTRACT FROM AUTHOR]

Summary: In the last few years, next‐generation sequencing techniques have started to be used to identify new viruses infecting plants. This has allowed to rapidly increase our knowledge on viruses other than those causing symptoms in economically important crops. Here we used this approach to identify a virus infecting Physcomitrium patens that has the typical structure of the double‐stranded RNA endogenous viruses of the Amalgaviridae family, which we named Physcomitrium patens amalgavirus 1, or PHPAV1. PHPAV1 is present only in certain accessions of P. patens, where its RNA can be detected throughout the cell cycle of the plant. Our analysis demonstrates that PHPAV1 can be vertically transmitted through both paternal and maternal germlines, in crosses between accessions that contain the virus with accessions that do not contain it. This work suggests that PHPAV1 can replicate in genomic backgrounds different from those that actually contain the virus and opens the door for future studies on virus–host coevolution. Significance Statement: We describe here Physcomitrium patens amalgavirus 1 (PHPAV1), the first virus associated with P. patens. PHPAV1 is a double‐stranded RNA endogenous virus of the Amalgaviridae family present in only certain accessions of P. patens and can be transmitted vertically through both paternal and maternal germlines. [ABSTRACT FROM AUTHOR]

The vast diversity of traits exhibited by horticultural crops largely depends upon variation in gene expression regulation. The uppermost layer of gene expression regulation is chromatin compaction. In plants, the LIKE HETEROCHROMATIN PROTEIN 1 (LHP1) is a member of the Polycomb Repressive Complex 1 (PRC1) that controls the spreading of the H3K27me3 mark throughout the genome to regulate gene expression. Much of the epigenetic control exerted by LHP1 has been deeply explored on the model species Arabidopsis thaliana. Recent advances in melon, tomato, and soybean highlight the relevance of LHP1 in controlling the development and physiology of a plethora of traits in crops. However, whether LHP1 exerts its diverse roles through similar mechanisms and through modulating the same target genes has been overlooked. In this review, we gather a wealth of knowledge about the LHP1 mode of action, which involves a tight connection with histone marks and long noncoding RNAs to modulate gene expression. Strikingly, we found that LHP1 may be linked to H3K27me3 regulation across the plant lineage, yet, through epigenetic regulation of a distinct set of target genes. This is supported by subtle differences in subcellular LHP1 localization between species found here. In addition, we summarize the variety of developmental outputs modulated by LHP1 across land plants pinpointing its importance for plant breeding. Hence, LHP1 has probably been co-opted in different lineages to modulate diverse traits contributing to crop diversification.

Targeted gene knockout is particularly useful for analyzing gene functions in plant growth, signaling, and development. By transforming knockout cassettes consisting of homologous sequences of the target gene into protoplasts, the classical gene targeting method aims to obtain targeted gene replacement, allowing for the characterization of gene functions in vivo. The moss Physcomitrella patens is a known model organism for a high frequency of homologous recombination and thus harbors a remarkable rate of gene targeting. Other moss features, including easy to culture, dominant haploidy phase, and sequenced genome, make gene targeting prevalent in Physcomitrella patens. However, even gene targeting was powerful to generate knockouts, researchers using this method still experienced technical challenges. For example, obtaining a good number of targeted knockouts after protoplast transformation and regeneration disturbed the users. Off-target mutations such as illegitimate random integration mediated by nonhomologous end joining and targeted insertion wherein one junction on-target but the other end off-target is commonly present in the knockouts. Protoplast fusion during transformation and regeneration was also a problem. This review will discuss the advantages and technical challenges of gene targeting. Recently, CRISPR-Cas9 is a revolutionary technology and becoming a hot topic in plant gene editing. In the second part of this review, CRISPR-Cas9 technology will be focused on and compared to gene targeting regarding the practical use in Physcomitrella patens. This review presents an updated perspective of the gene targeting and CRISPR-Cas9 techniques to plant biologists who may consider studying gene functions in the model organism Physcomitrella patens.

Key message: Homologous genes for the peptidoglycan precursor flippase MurJ, and peptidoglycan hydrolases: lytic transglycosylase MltB, and dd-carboxypeptidase VanY are required for chloroplast division in the moss Physcomitrella patens. The moss Physcomitrella patens is used as a model plant to study plastid peptidoglycan biosynthesis. In bacteria, MurJ flippase transports peptidoglycan precursors from the cytoplasm to the periplasm. In this study, we identified a MurJ homolog (PpMurJ) in the P. patens genome. Bacteria employ peptidoglycan degradation and recycling pathways for cell division. We also searched the P. patens genome for genes homologous to bacterial peptidoglycan hydrolases and identified genes homologous for the lytic transglycosylase mltB, N-acetylglucosaminidase nagZ, and ld-carboxypeptidase ldcA in addition to a putative dd-carboxypeptidase vanY reported previously. Moreover, we found a ß-lactamase-like gene (Pplactamase). GFP fusion proteins with either PpMltB or PpVanY were detected in the chloroplasts, whereas fusion proteins with PpNagZ, PpLdcA, or Pplactamase localized in the cytoplasm. Experiments seeking PpMurJ-GFP fusion proteins failed. PpMurJ gene disruption in P. patens resulted in the appearance of macrochloroplasts in protonemal cells. Compared with the numbers of chloroplasts in wild-type plants (38.9 ± 4.9), PpMltB knockout and PpVanY knockout had lower numbers of chloroplasts (14.3 ± 6.7 and 28.1 ± 5.9, respectively). No differences in chloroplast numbers were observed after PpNagZ, PpLdcA, or Pplactamase single-knockout. Chloroplast numbers in PpMltB/PpVanY double-knockout cells were similar to those in PpMltB single-knockout cells. Zymogram analysis of the recombinant PpMltB protein revealed its peptidoglycan hydrolase activity. Our results imply that PpMurJ, PpMltB and PpVanY play a critical role in chloroplast division in the moss P. patens. [ABSTRACT FROM AUTHOR]

SOG1 is a crucial plant-specific NAC domain family transcription factor and functions as the central regulator of DNA damage response, acting downstream of ATM and ATR kinases. In this study, various in-silico approaches have been employed for the characterization of SOG1 transcription factor in a comparative manner with its orthologues from various plant species. Amino acid sequences of more than a hundred SOG1 or SOG1-like proteins were retrieved and their relationship was determined through phylogenetic and motif analyses. Various physiochemical properties and secondary structural components of SOG1 orthologues were determined in selective plant species including Arabidopsis thaliana , Oryza sativa , Amborella trichopoda , and Physcomitrella patens. Furthermore, fold recognition or threading and homology-based three-dimensional models of SOG1 were constructed followed by subsequent evaluation of quality and accuracy of the generated protein models. Finally, extensive DNA-Protein and Protein-Protein interaction studies were performed using the HADDOCK server to give an insight into the mechanism of how SOG1 binds with the promoter region of its target genes or interacts with other proteins to regulate the DNA damage responses in plants. Our docking analysis data have shown the molecular mechanism of SOG1′s binding with 5′-CTT(N) 7 AAG-3′ and 5′-(N) 4 GTCAA(N) 4 -3′ consensus sequences present in the promoter region of its target genes. Moreover, SOG1 physically interacts and forms a thermodynamically stable complex with NAC103 and BRCA1 proteins, which possibly serve as coactivators or mediators in the transcription regulatory network of SOG1. Overall, our in-silico study will provide meaningful information regarding the structural and functional characterization of the SOG1 transcription factor. [ABSTRACT FROM AUTHOR]

2D or not 2D: how mRNA methylation regulates the transition to 3-dimensional growth in Physcomitrium patens Once they had these viable mutants with aberrant m SP 6 sp A contents, they screened them for mutant phenotypes and found that the mutants displayed delayed gametophore bud formation, meaning that they had shown m SP 6 sp A modifications are integral for the transition from 2-dimensional to 3-dimensional growth. To regulate growth, development, and responses to the environment properly, organisms have evolved a plethora of mechanisms for exerting tight control over the expression of their genes. [Extracted from the article]

S1-like nucleases are widespread enzymes commonly used in biotechnology and molecular biology. Although it is commonly believed that they are mainly Zn2+-dependent acidic enzymes, we have found that numerous members of this family deviate from this rule. Therefore, in this work, we decided to check how broad is the range of non‑zinc-dependent S1-like nucleases and what is the molecular basis of their activities. S1-like nucleases chosen for analysis were achieved through heterologous expression in appropriate eukaryotic hosts. To characterize nucleases' active-site properties, point mutations were introduced in selected positions. The enzymatic activities of wild-type and mutant nucleases were tested by in-gel nuclease activity assay. We discovered that S1-like nucleases encoded by non-vascular plants and single-celled protozoa, like their higher plant homologues, exhibit a large variety of catalytic properties. We have shown that these individual properties are determined by specific non-conserved active site residues. Our findings demonstrate that mutations that occur during evolution can significantly alter the catalytic properties of S1-like nucleases. As a result, different ions can compete for particular S1-type nucleases' active sites. This phenomenon undermines the existing classification of S1-like nucleases. Our findings have numerous implications for applications and understanding the S1-like nucleases' biological functions. For example, new biotechnological applications should take into account their unexpected catalytic properties. Moreover, these results demonstrate that the trinuclear zinc-based model commonly used to characterize the catalytic activities of S1-like nucleases is insufficient to explain the actions of non‑zinc-dependent members of this family. • The class of non‑zinc-dependent S1-like nucleases is widely distributed among different phylogenetic groups. • The active sites of S1-like nucleases are highly vulnerable to changes in activity. • The individual requirements of S1-like nucleases are determined by the composition of non-conserved active site residues. • S1-like nuclease family members can adapt to varied physiological conditions and thus contribute to a variety of functions. • The biotechnological applications of S1-like nucleases must take into account the wide range of their activities. [ABSTRACT FROM AUTHOR]

Plant oxylipins are a class of lipid-derived signaling molecules being involved in the regulation of various biotic and abiotic stress responses. A major class of oxylipins are the circular derivatives to which 12-oxo-phytodienoic acid (OPDA) and its metabolite jasmonic acid (JA) belong. While OPDA and its shorter chain homologue dinor -OPDA (dnOPDA) seem to be ubiquitously found in land plants ranging from bryophytes to angiosperms, the occurrence of JA and its derivatives is still under discussion. The bryophyte Physcomitrium patens has received increased scientific interest as a non-vascular plant model organism over the last decade. Therefore, we followed the metabolism upon wounding by metabolite fingerprinting with the aim to identify jasmonates as well as novel oxylipins in P. patens . A non-targeted metabolomics approach was used to reconstruct the metabolic pathways for the synthesis of oxylipins, derived from roughanic, linoleic, α-linolenic, and arachidonic acid in wild type, the oxylipin-deficient mutants of Ppaos1 and Ppaos2 , the mutants of Ppdes being deficient in all fatty acids harboring a Δ 6 -double bond and the C20-fatty acid-deficient mutants of Ppelo . Beside of OPDA, iso -OPDA, dnOPDA, and iso -dnOPDA, three additional C18-compounds and a metabolite being isobaric to JA were identified to accumulate after wounding. These findings can now serve as foundation for future research in determining, which compound(s) will serve as native ligand(s) for the oxylipin-receptor COI1 in P. patens ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Resemann, Feussner, Hornung and Feussner.)

Plant and animal stem cells can self-renew and give rise to differentiated cells to form tissues or organs. Unlike differentiated cells in animals, those in land plants can be readily reprogrammed into stem cells, reflecting the plasticity of plant cell identity. The moss Physcomitrium patens (synonym: Physcomitrella patens) is highly regenerable, and its leaf cells can be reprogrammed into stem cells in response to wounding or by transient DNA damage without wounding. Wounding and DNA damage induce STEM CELL-INDUCING FACTOR 1 , an APETALA2/ETHYLENE RESPONSE FACTOR. Here, we summarize the genetic networks that regulate cellular reprogramming in P. patens and the roles of STEMIN1 and discuss the generality and divergence of the molecular mechanisms underlying cellular reprogramming in land plants and animals. [Display omitted] • Some types of cells including terminally differentiated cells in land plants can be reprogrammed into stem cells by wounding. • Wounding and DNA damage change Physcomitrium (synonym: Physcomitrella) patens leaf cells into chloronema apical stem cells. • S TEMIN , PpWOX13L , PpCSP and ATG genes form a regulatory network of wound-induced reprogramming in P. patens. • CSPs with putative RNA-binding activity are involved in reprogramming into stem cells in both mammals and land plants. • Stem cell formation by wounding and DNA damage should be adaptive for land plants to survive under a harsh environment. [ABSTRACT FROM AUTHOR]

Land colonization is a major event in plant evolution. Little is known about the evolutionary characteristics of lipids during this process. Here, we proved that Physcomitrella patens , a bryophyte that appeared in the early evolution of terrestrial plants, has short-term desiccation resistance. The maintenance of membrane integrity is related to its specific glycerolipid composition and key genes for lipid metabolism. We analyzed 414 types of lipid molecules, and found that phospholipids accounted for 61.7%, mainly PC and PI; glycolipids accounted for only 26.5%, with a special MGDG molecular map. The most abundant MDGD, that is, MGDG34:6, contained rare 15- and 19-carbon acyl chains; the level of neutral lipids was higher. This was consistent with the results observed by TEM, with fewer lamellae and obvious lipid droplets. Slight dehydration accumulated a large number of TAG molecules, and severe dehydration degraded phospholipids and caused membrane leakage, but PA and MGDG fluctuated less. The key genes of lipid metabolism, DGAT and PAP , were actively transcribed, suggesting that PA was one of the main DAG sources for TAG synthesis. This work proves that Physcomitrella patens adopts high-constitutive PC and PI similar to plant seeds, abundant TAG, and its own specific MGDG to resist extreme dehydration. This result provides a new insight into the lipid evolution of early terrestrial plants against unfavorable terrestrial environments. [Display omitted] • Short-term desiccation resistance naturally exists in Physcomitrella patens. • Highly constitutive PC and PI similar to plant seeds. • Specific MGDG map, the most dominant lipid species contains two odd-numbered acyl chains. • Rapid dehydration accumulates TAG and stimulates the pathway from PA to DAG. • The specific glycerolipid composition establishes the dehydration tolerance of early land plants. [ABSTRACT FROM AUTHOR]

To survive in the dry state, orthodox seeds acquire desiccation tolerance. As maturation progresses, the seeds gradually acquire longevity, which is the total timespan during which the dry seeds remain viable. The desiccation-tolerance mechanism(s) allow seeds to remain dry without losing their ability to germinate. This adaptive trait has played a key role in the evolution of land plants. Understanding the mechanisms for seed survival after desiccation is one of the central goals still unsolved. That is, the cellular protection during dry state and cell repair during rewatering involves a not entirely known molecular network(s). Although desiccation tolerance is retained in seeds of higher plants, resurrection plants belonging to different plant lineages keep the ability to survive desiccation in vegetative tissue. Abscisic acid (ABA) is involved in desiccation tolerance through tight control of the synthesis of unstructured late embryogenesis abundant (LEA) proteins, heat shock thermostable proteins (sHSPs), and non-reducing oligosaccharides. During seed maturation, the progressive loss of water induces the formation of a so-called cellular "glass state". This glassy matrix consists of soluble sugars, which immobilize macromolecules offering protection to membranes and proteins. In this way, the secondary structure of proteins in dry viable seeds is very stable and remains preserved. ABA insensitive-3 (ABI3), highly conserved from bryophytes to Angiosperms, is essential for seed maturation and is the only transcription factor (TF) required for the acquisition of desiccation tolerance and its re-induction in germinated seeds. It is noteworthy that chlorophyll breakdown during the last step of seed maturation is controlled by ABI3. This update contains some current results directly related to the physiological, genetic, and molecular mechanisms involved in survival to desiccation in orthodox seeds. In other words, the mechanisms that facilitate that an orthodox dry seed is a living entity. [ABSTRACT FROM AUTHOR]

• Terpene production in three bryophyte species under salt stress was documented. • Histochemical compartmentalization of terpenes in the mosses was recorded. • Terpenoid compounds are mainly presented in the protonemal cells of all tested bryophyte species. • Terpenes could also be found in the phylloids, which is in correlation with NaCl stress intensity. • Exogenously added ABA induced higher terpenoid production in two funaroid moss species, but lower production of terpenoid compounds and different terpenes composition in the pottioid moss. Histochemical analyses were applied to document the presence of terpenoids in three selected mosses. The species were chosen as bryo-halophyte, namely Entosthodon hungaricus (Funariaceae) and Hennediella heimii (Pottiaceae) and a model moss known to possess salt tolerance, Physcomitrella patens (Funariaceae). They were exposed to sodium chloride under axenic in vitro fully controlled conditions, with the aim to compare the terpenoid evidences among different moss developmental stages and species in various experimental treatments. The stress was simulated by addition of NaCl to the growth medium but also exogenously added ABA simulating the plantlets adjustment to increasing stress. The results obtained unequivocally indicate that the selected mosses respond to exogenous ABA as well as to NaCl in a different manner as indicated by histochemical staining of terpenes. Additionally, terpenoids were confirmed by colorimetric tests and in accordance with histochemical analyses. ABA seems to play role in pathways of terpenoid production in the two funaroid mosses, at least indirectly, implying these features to be evolutionary early developed in common funaroid ancestor. In contrast, the pottioid moss H. heimii differed in its response to stress by the constitutive presence of terpenoid compounds in all stress treatments. [ABSTRACT FROM AUTHOR]

In bryophytes (i.e., mosses, liverworts, and hornworts), extant representatives of early land plants, plasmodesmata have been described in a wide range of tissues. Although their contribution to bryophyte morphogenesis remains largely unexplored, several recent studies have suggested that the deposition of callose around plasmodesmata might regulate developmental and physiological responses in mosses. In this chapter, we provide a protocol to image and quantify callose levels in the filamentous body of the model moss Physcomitrium (Physcomitrella) patens and discuss possible alternatives and pitfalls. More generally, this protocol establishes a framework to explore the distribution of callose in other bryophytes., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Protoplast production with the moss Physcomitrium (Physcomitrella) patens has a long and successful history. As a tool, it has not only been the base of reverse genetic studies covering research fields as diverse as development, metabolism, or gene network regulation but also allowed its development as a bioengineering platform for protein production. We present here a standardized protocol for protoplast production from Physcomitrium (Physcomitrella) patens protonemata. Additionally, we detail procedures for their transfection, their plating for optimal regeneration, and three alternative selection approaches. To improve the consistency of protoplast regeneration, we describe a new option for protoplast embedding. The use of an alginate matrix to regenerate moss protoplast alleviates the use of warm agarized medium. Thus, it optimizes transformed protoplast survival without any morphological detrimental effect or impact on transfection efficiency., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

To survive in the dry state, orthodox seeds acquire desiccation tolerance. As maturation progresses, the seeds gradually acquire longevity, which is the total timespan during which the dry seeds remain viable. The desiccation-tolerance mechanism(s) allow seeds to remain dry without losing their ability to germinate. This adaptive trait has played a key role in the evolution of land plants. Understanding the mechanisms for seed survival after desiccation is one of the central goals still unsolved. That is, the cellular protection during dry state and cell repair during rewatering involves a not entirely known molecular network(s). Although desiccation tolerance is retained in seeds of higher plants, resurrection plants belonging to different plant lineages keep the ability to survive desiccation in vegetative tissue. Abscisic acid (ABA) is involved in desiccation tolerance through tight control of the synthesis of unstructured late embryogenesis abundant (LEA) proteins, heat shock thermostable proteins (sHSPs), and non-reducing oligosaccharides. During seed maturation, the progressive loss of water induces the formation of a so-called cellular "glass state". This glassy matrix consists of soluble sugars, which immobilize macromolecules offering protection to membranes and proteins. In this way, the secondary structure of proteins in dry viable seeds is very stable and remains preserved. ABA insensitive-3 (ABI3), highly conserved from bryophytes to Angiosperms, is essential for seed maturation and is the only transcription factor (TF) required for the acquisition of desiccation tolerance and its re-induction in germinated seeds. It is noteworthy that chlorophyll breakdown during the last step of seed maturation is controlled by ABI3. This update contains some current results directly related to the physiological, genetic, and molecular mechanisms involved in survival to desiccation in orthodox seeds. In other words, the mechanisms that facilitate that an orthodox dry seed is a living entity.

Targeted gene knockout is particularly useful for analyzing gene functions in plant growth, signaling, and development. By transforming knockout cassettes consisting of homologous sequences of the target gene into protoplasts, the classical gene targeting method aims to obtain targeted gene replacement, allowing for the characterization of gene functions in vivo . The moss Physcomitrella patens is a known model organism for a high frequency of homologous recombination and thus harbors a remarkable rate of gene targeting. Other moss features, including easy to culture, dominant haploidy phase, and sequenced genome, make gene targeting prevalent in Physcomitrella patens . However, even gene targeting was powerful to generate knockouts, researchers using this method still experienced technical challenges. For example, obtaining a good number of targeted knockouts after protoplast transformation and regeneration disturbed the users. Off-target mutations such as illegitimate random integration mediated by nonhomologous end joining and targeted insertion wherein one junction on-target but the other end off-target is commonly present in the knockouts. Protoplast fusion during transformation and regeneration was also a problem. This review will discuss the advantages and technical challenges of gene targeting. Recently, CRISPR-Cas9 is a revolutionary technology and becoming a hot topic in plant gene editing. In the second part of this review, CRISPR-Cas9 technology will be focused on and compared to gene targeting regarding the practical use in Physcomitrella patens . This review presents an updated perspective of the gene targeting and CRISPR-Cas9 techniques to plant biologists who may consider studying gene functions in the model organism Physcomitrella patens ., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zhu.)