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Group II introns are large catalytic RNAs, which reside mainly within genes encoding respiratory complex I (CI) subunits in angiosperms' mitochondria. Genetic and biochemical analyses led to the identification of many nuclear-encoded factors that facilitate the splicing of the degenerated organellar introns in plants. Here, we describe the analysis of the pentatricopeptide repeat (PPR) co-expressed intron splicing-1 (PCIS1) factor, which was identified in silico by its co-expression pattern with many PPR proteins. PCIS1 is well conserved in land plants but has no sequence similarity with any known protein motifs. PCIS1 mutant lines are arrested in embryogenesis and can be maintained by the temporal expression of the gene under the embryo-specific ABI3 promoter. The p ABI3::PCIS1 mutant plants display low germination and stunted growth phenotypes. RNA-sequencing and quantitative RT-PCR analyses of wild-type and mutant plants indicated that PCIS1 is a novel splicing cofactor that is pivotal for the maturation of several nad transcripts in Arabidopsis mitochondria. These phenotypes are tightly associated with respiratory CI defects and altered plant growth. Our data further emphasize the key roles of nuclear-encoded cofactors that regulate the maturation and expression of mitochondrial transcripts for the biogenesis of the oxidative phosphorylation system, and hence for plant physiology. The discovery of novel splicing factors other than typical RNA-binding proteins suggests further complexity of splicing mechanisms in plant mitochondria. [ABSTRACT FROM AUTHOR]

Thermospermine suppresses auxin-inducible xylem differentiation, whereas its structural isomer, spermine, is involved in stress responses in angiosperms. The thermospermine synthase, ACAULIS5 (ACL5), is conserved from algae to land plants, but its physiological functions remain elusive in non-vascular plants. Here, we focused on Mp ACL5 , a gene in the liverwort Marchantia polymorpha , that rescued the dwarf phenotype of the acl5 mutant in Arabidopsis. In the Mp acl5 mutants generated by genome editing, severe growth retardation was observed in the vegetative organ, thallus, and the sexual reproductive organ, gametangiophore. The mutant gametangiophores exhibited remarkable morphological defects such as short stalks, fasciation and indeterminate growth. Two gametangiophores fused together, and new gametangiophores were often initiated from the old ones. Furthermore, Mp acl5 showed altered responses to heat and salt stresses. Given the absence of spermine in bryophytes, these results suggest that thermospermine has a dual primordial function in organ development and stress responses in M. polymorpha. The stress response function may have eventually been assigned to spermine during land plant evolution. [ABSTRACT FROM AUTHOR]

Sexual differentiation is a fundamental process in the life cycles of land plants, ensuring successful sexual reproduction and thereby contributing to species diversity and survival. In the dioicous liverwort Marchantia polymorpha , this process is governed by an autosomal sex-differentiation locus comprising FEMALE GAMETOPHYTE MYB (FGMYB), a female-promoting gene, and SUPPRESSOR OF FEMINIZATION (SUF), an antisense strand-encoded long non-coding RNA (lncRNA). SUF is specifically transcribed in male plants and suppresses the expression of FGMYB , leading to male differentiation. However, the molecular mechanisms underlying this process remain elusive. Here, we show that SUF acts through its transcription to suppress FGMYB expression. Transgene complementation analysis using CRISPR/Cas9D10A-based large-deletion mutants identified a genomic region sufficient for the sex differentiation switch function in the FGMYB-SUF locus. Inserting a transcriptional terminator sequence into the SUF- transcribed region resulted in the loss of SUF function and allowed expression of FGMYB in genetically male plants, leading to conversion of the sex phenotype from male to female. Partial deletions of SUF had no obvious impact on its function. Replacement of the FGMYB sequence with that of an unrelated gene did not affect the ability of SUF transcription to suppress sense-strand expression. Taken together, our findings suggest that the process of SUF transcription, rather than the resulting transcripts, is required for controlling sex differentiation in M. polymorpha. [ABSTRACT FROM AUTHOR]

In land plants, sexual dimorphism can develop in both diploid sporophytes and haploid gametophytes. While developmental processes of sexual dimorphism have been extensively studied in the sporophytic reproductive organs of model flowering plants such as stamens and carpels of Arabidopsis thaliana , those occurring in gametophyte generation are less well characterized due to the lack of amenable model systems. In this study, we performed three-dimensional morphological analyses of gametophytic sexual branch differentiation in the liverwort Marchantia polymorpha, using high-depth confocal imaging and a computational cell segmentation technique. Our analysis revealed that the specification of germline precursors initiates in a very early stage of sexual branch development, where incipient branch primordia are barely recognizable in the apical notch region. Moreover, spatial distribution patterns of germline precursors differ between males and females from the initial stage of primordium development in a manner dependent on the master sexual differentiation regulator Mp FGMYB. At later stages, distribution patterns of germline precursors predict the sex-specific gametangia arrangement and receptacle morphologies seen in mature sexual branches. Taken together, our data suggest a tightly coupled progression of germline segregation and sexual dimorphism development in M. polymorpha. [ABSTRACT FROM AUTHOR]

The liverwort Marchantia polymorpha is equipped with a wide range of molecular and genetic tools and resources that have led to its wide use to explore the evo-devo aspects of land plants. Although its diverse transcriptome data are rapidly accumulating, there is no extensive yet user-friendly tool to exploit such a compilation of data and to summarize results with the latest annotations. Here, we have developed a web-based suite of tools, MarpolBase Expression (MBEX, https://marchantia.info/mbex/), where users can visualize gene expression profiles, identify differentially expressed genes, perform co-expression and functional enrichment analyses and summarize their comprehensive output in various portable formats. Using oil body biogenesis as an example, we demonstrated that the results generated by MBEX were consistent with the published experimental evidence and also revealed a novel transcriptional network in this process. MBEX should facilitate the exploration and discovery of the genetic and functional networks behind various biological processes in M. polymorpha and promote our understanding of the evolution of land plants. [ABSTRACT FROM AUTHOR]

Regeneration in land plants is accompanied by the establishment of new stem cells, which often involves reactivation of the cell division potential in differentiated cells. The phytohormone auxin plays pivotal roles in this process. In bryophytes, regeneration is enhanced by the removal of the apex and repressed by exogenously applied auxin, which has long been proposed as a form of apical dominance. However, the molecular basis behind these observations remains unexplored. Here, we demonstrate that in the liverwort Marchantia polymorpha , the level of endogenous auxin is transiently decreased in the cut surface of decapitated explants, and identify by transcriptome analysis a key transcription factor gene, LOW- AUXIN RESPONSIVE (Mp LAXR), which is induced upon auxin reduction. Loss of Mp LAXR function resulted in delayed cell cycle reactivation, and transient expression of Mp LAXR was sufficient to overcome the inhibition of regeneration by exogenously applied auxin. Furthermore, ectopic expression of Mp LAXR caused cell proliferation in normally quiescent tissues. Together, these data indicate that decapitation causes a reduction of auxin level at the cut surface, where, in response, Mp LAXR is up-regulated to trigger cellular reprogramming. Mp LAXR is an ortholog of Arabidopsis ENHANCER OF SHOOT REGENERATION 1/DORNR Ö SCHEN , which has dual functions as a shoot regeneration factor and a regulator of axillary meristem initiation, the latter of which requires a low auxin level. Thus, our findings provide insights into stem cell regulation as well as apical dominance establishment in land plants. [ABSTRACT FROM AUTHOR]

Regulation of the stability and the quality of mitochondrial RNA is essential for the maintenance of mitochondrial and cellular functions in eukaryotes. We have previously reported that the eukaryotic poly(A)-specific ribonuclease (PARN) and the prokaryotic poly(A) polymerase encoded by AHG2 and AGS1 , respectively, coordinately regulate the poly(A) status and the stability of mitochondrial mRNA in Arabidopsis. Mitochondrial function of PARN has not been reported in any other eukaryotes. To know how much this PARN-based mitochondrial mRNA regulation is conserved among plants, we studied the AHG2 and AGS1 counterparts of the liverwort, Marchantia polymorpha , a member of basal land plant lineage. We found that M. polymorpha has one ortholog each for AHG2 and AGS1 , named Mp AHG2 and Mp AGS1 , respectively. Their Citrine-fused proteins were detected in mitochondria of the liverwort. Molecular genetic analysis showed that Mp AHG2 is essential and functionally interacts with Mp AGS1 as observed in Arabidopsis. A recombinant MpAHG2 protein had a deadenylase activity in vitro. Overexpression of Mp AGS1 and the reduced expression of Mp AHG2 caused an accumulation of polyadenylated Mp cox1 mRNA. Furthermore, Mp AHG2 suppressed Arabidopsis ahg2-1 mutant phenotype. These results suggest that the PARN-based mitochondrial mRNA regulatory system is conserved in land plants. [ABSTRACT FROM AUTHOR]

Mitochondria are dynamic organelles that have inner and outer membranes. In plants, the inner membrane has been well studied but relatively little is known about the outer membrane. Here we report that Arabidopsis cells have mitochondrial outer membrane-derived structures, some of which protrude from the main body of mitochondria (itochondrial uter-membrane rotrusions; MOPs), while others form vesicle-like structures without a matrix marker. The latter vesicle-like structures are similar to some mammalian MDVs (itochondrial-erived esicles). Live imaging demonstrated that a plant MDV budded off from the tip of a MOP. MDVs were also observed in the drp3a drp3b double mutant, indicating that they could be formed without the mitochondrial fission factors DRP3A and DRP3B. Double staining studies showed that the MDVs were not peroxisomes, endosomes, Golgi apparatus or trans-Golgi network (TGN). The numbers of MDVs and MOPs increased in senescent leaves and after dark treatment. Together, these results suggest that MDVs and MOPs are related to leaf senescence. [ABSTRACT FROM AUTHOR]

Engagement of the Fas receptor (CD95) initiates multiple signaling pathways that lead to apoptosis, such as the formation of death-inducing signaling complex (DISC), activation of caspase cascades, and the generation of the lipid messenger, ceramide. Sphingomyelin (SM) is a major component of lipid rafts, which are specialized structures that enhance the efficiency of membrane receptor signaling and are a main source of ceramide. However, the functions of SM in Fas-mediated apoptosis have yet to be clearly defined, as the responsible genes have not been identified. After cloning a gene responsible for SM synthesis, SMS1, we established SM synthase-defective WR19L cells transfected with the human Fas gene (WR/Fas-SM(-)), and cells that have been functionally restored by transfection with SMS1 (WR/Fas-SMS1). We show that expression of membrane SM enhances Fas-mediated apoptosis through increasing DISC formation, activation of caspases, efficient translocation of Fas into lipid rafts, and subsequent Fas clustering. Furthermore, WR/Fas-SMS1 cells, but not WR/Fas-SM(--) cells, showed a considerable increase in ceramide generation within lipid rafts upon Fas stimulation. These data suggest that a membrane SM is important for Fas clustering through aggregation of lipid rafts, leading to Fas-mediated apoptosis. [ABSTRACT FROM AUTHOR]

Sphingomyelin (SM) synthase has been assumed to be involved in both cell death and survival by regulating pro-apoptotic mediator ceramide and pro-survival mediator diacylglycerol. However, its precise functions are ambiguous due to the lack of molecular cloning of SM synthase gene(s). We isolated WR19L/Fas-SM(-) mouse lymphoid cells, which show a defect of SM at the plasma membrane due to the lack of SM synthase activity and resistance to cell death induced by an SM-directed cytolytic protein lysenin. WR19L/Fas-SM(-) cells were also highly susceptible to methyl-β-cyclodextrin (MβCD) as compared with the WR19L/Fas-SM(+) cells, which are capable of SM synthesis. By expression cloning method using WR19L/Fas-SM(-) cells and MβCD-based selection, we have succeeded in cloning of a human cDNA responsible for SM synthase activity. The cDNA encodes a peptide of 413 amino acids named SMS1 (putative molecular mass, 48.6 kDa), which contains a sterile a motif domain near the N-terminal region and four predicted transmembrane domains. WR19L/Fas-SM(-) cells expressing SMS1 cDNA (WR19L/Fas-SMS1) restored the resistance against MβCD, the accumulation of SM at the plasma membrane, and SM synthesis by transferring phosphocholine from phosphatidylcholine to ceramide. Furthermore, WR19L/Fas-SMS1 cells, as well as WR19L/Fas-SM(-) cells supplemented with exogenous SM, restored cell growth ability in serum-free conditions, where the growth of WR19L/Fas-SM(-) cells was severely inhibited. The results suggest that SMS1 is responsible for SM synthase activity in mammalian cells and plays a critical role in cell growth of mouse lymphoid cells. [ABSTRACT FROM AUTHOR]

Presents a functional analysis of a beta-ketoacyl-CoA synthase (KCS) gene, MpFAE2, by gene silencing in the liverwort Marchantia polymorpha L. Isolation of a KCS gene homologue, MpFAE2, from M. polymorpha; Characterization of the putative MpFAE2 product; Transgenic M. polymorpha plants for gene silencing and overexpression of the MpFAE2 gene.

Summary Land plants differentiate germ cells in the haploid gametophyte. In flowering plants, a generative cell is specified as a precursor that subsequently divides into two sperm cells in the developing male gametophyte, pollen. Generative cell specification requires cell-cycle control and microtubule-dependent nuclear relocation (reviewed in [ 1–3 ]). However, the generative cell fate determinant and its evolutionary origin are still unknown. In bryophytes, gametophytes produce eggs and sperm in multicellular reproductive organs called archegonia and antheridia, respectively, or collectively called gametangia. Given the monophyletic origin of land plants [ 4–6 ], evolutionarily conserved mechanisms may play key roles in these diverse reproductive processes. Here, we showed that a single member of the subfamily VIIIa of basic helix-loop-helix (bHLH) transcription factors in the liverwort Marchantia polymorpha primarily accumulated in the initial cells and controlled their development into gametangia. We then demonstrated that an Arabidopsis thaliana VIIIa bHLH transiently accumulated in the smaller daughter cell after an asymmetric division of the meiosis-derived microspore and was required for generative cell specification redundantly with its paralog. Furthermore, these A. thaliana VIIIa bHLHs were functionally replaceable by the M. polymorpha VIIIa bHLH. These findings suggest the VIIIa bHLH proteins as core regulators for reproductive development, including germ cell differentiation, since an early stage of land plant evolution. [ABSTRACT FROM AUTHOR]

Among the eukaryotes only plants and a number of fungi are able to synthesize biotin. Although initial events leading to the biosynthesis of biotin remain largely unknown, the final steps are known to occur in the mitochondria. Here we deleted the Aopex5 and Aopex7 genes encoding the receptors for peroxisomal targeting signals PTS1 and PTS2, respectively, in the filamentous fungus Aspergillus oryzae. In addition to exhibiting defects in the peroxisomal targeting of either PTS1 or PTS2 proteins, the deletion strains also displayed growth defects on minimal medium containing oleic acid as the sole carbon source. Unexpectedly, these peroxisomal transport-deficient strains also exhibited growth defects on minimal medium containing glucose as the sole carbon source that were remediated by the addition of biotin and its precursors, including 7-keto-8-aminopelargonic acid (KAPA). Genome database searches in fungi and plants revealed that BioF protein/KAPA synthase, one of the biotin biosynthetic enzymes, has a PTS1 sequence at the C terminus. Fungal ΔbioF strains expressing the fungal and plant BioF proteins lacking PTS1 still exhibited growth defects in the absence of biotin, indicating that peroxisomal targeting of KAPA synthase is crucial for the biotin biosynthesis. Furthermore, in the plant Arabidopsis thaliana, AtBioF localized to the peroxisomes through recognition of its PTS1 sequence, suggesting involvement of peroxisomes in biotin biosynthesis in plants. Taken together we demonstrate a novel role for peroxisomes in biotin biosynthesis and suggest the presence of as yet unidentified peroxisomal proteins that function in the earlier steps of biotin biosynthesis. [ABSTRACT FROM AUTHOR]

Sphingomyelin synthase 1 (SMS1) catalyzes the conversion of ceramide to sphingomyelin. Here, we generated and analyzed SMS 1-null mice. SMS1-null mice exhibited moderate neonatal lethality, reduced body weight, and loss of fat tissues mass, suggesting that they might have metabolic abnormality. Indeed, analysis on glucose metabolism revealed that they showed severe deficiencies in insulin secretion. Isolated mutant islets exhibited severely impaired ability to release insulin, dependent on glucose stimuli. Further analysis indicated that mitochondria in mutant islet cells cannot up-regulate ATP production in response to glucose. We also observed additional mitochondrial abnormalities, such as hyperpolarized membrane potential and increased levels of reactive oxygen species (ROS) in mutant islets. Finally, when SMS1-null mice were treated with the anti-oxidant N-acetyl cysteine, we observed partial recovery of insulin secretion, indicating that ROS overproduction underlies pancreatic β-cell dysfunction in SMS1-null mice. Altogether, our data suggest that SMS1 is important for controlling ROS generation, and that SMS1 is required for normal mitochondrial function and insulin secretion in pancreatic β-cells. [ABSTRACT FROM AUTHOR]

Sex determination is a central process for sexual reproduction and is often regulated by a sex determinant encoded on a sex chromosome. Rules that govern the evolution of sex chromosomes via specialization and degeneration following the evolution of a sex determinant have been well studied in diploid organisms. However, distinct predictions apply to sex chromosomes in organisms where sex is determined in the haploid phase of the life cycle: both sex chromosomes, female U and male V, are expected to maintain their gene functions, even though both are non-recombining. This is in contrast to the X-Y (or Z-W) asymmetry and Y (W) chromosome degeneration in XY (ZW) systems of diploids. Here, we provide evidence that sex chromosomes diverged early during the evolution of haploid liverworts and identify the sex determinant on the Marchantia polymorpha U chromosome. This gene, Feminizer , encodes a member of the plant-specific BASIC PENTACYSTEINE transcription factor family. It triggers female differentiation via regulation of the autosomal sex-determining locus of FEMALE GAMETOPHYTE MYB and SUPPRESSOR OF FEMINIZATION. Phylogenetic analyses of Feminizer and other sex chromosome genes indicate dimorphic sex chromosomes had already been established 430 mya in the ancestral liverwort. Feminizer also plays a role in reproductive induction that is shared with its gametolog on the V chromosome, suggesting an ancestral function, distinct from sex determination, was retained by the gametologs. This implies ancestral functions can be preserved after the acquisition of a sex determination mechanism during the evolution of a dominant haploid sex chromosome system. [Display omitted] • Feminizer (BPCU) linked to the liverwort female sex chromosome was identified • BPCU regulates the autosomal sex-determining locus, FGMYB /S UF • BPCU additionally shares a function for reproductive induction with its gametolog • The liverwort sex chromosomes are the oldest among any other known systems Iwasaki et al. identify a female sex determinant, Feminizer , on the female sex chromosome of a haploid plant, Marchantia polymorpha. Unexpectedly, Feminizer also plays a role in reproductive induction, which is shared by its male gametolog. The liverwort sex chromosomes diverged around 430 mya, older than any known sex chromosome systems. [ABSTRACT FROM AUTHOR]

Plant survival depends on the optimal use of resources under variable environmental conditions. Among the mechanisms that mediate the balance between growth, differentiation, and stress responses, the regulation of transcriptional activity by DELLA proteins stands out. In angiosperms, DELLA accumulation promotes defense against biotic and abiotic stress and represses cell division and expansion, while the loss of DELLA function is associated with increased plant size and sensitivity toward stress. 1 Given that DELLA protein stability is dependent on gibberellin (GA) levels 2 and GA metabolism is influenced by the environment, 3 this pathway is proposed to relay environmental information to the transcriptional programs that regulate growth and stress responses in angiosperms. 4,5 However, DELLA genes are also found in bryophytes, whereas canonical GA receptors have been identified only in vascular plants. 6–10 Thus, it is not clear whether these regulatory functions of DELLA predated or emerged with typical GA signaling. Here, we show that, as in vascular plants, the only DELLA in the liverwort Marchantia polymorpha also participates in the regulation of growth and key developmental processes and promotes oxidative stress tolerance. Moreover, part of these effects is likely caused by the conserved physical interaction with the MpPIF transcription factor. Therefore, we suggest that the role in the coordination of growth and stress responses was already encoded in the DELLA protein of the common ancestor of land plants, and the importance of this function is underscored by its conservation over the past 450 million years. [Display omitted] • MpDELLA accumulation impairs thallus growth and cell division • MpDELLA interacts with MpPIF to modulate developmental traits • Mp DELLA expression increases under oxidative stress and promotes stress tolerance • Coordination between growth and stress responses by DELLA predates GA signaling DELLA proteins coordinate growth and stress responses as part of gibberellin signaling in angiosperms. Hernández-García, Sun, et al. show that this role is also conserved in Marchantia polymorpha despite the lack of a bona fide gibberellin perception module in bryophytes. [ABSTRACT FROM AUTHOR]

In angiosperms, the phase transition from vegetative to reproductive growth involves the de-repression of the squamosa promoter-binding-protein-like (SPL) class of transcription factors, which is negatively regulated by the specific microRNAs (miRNAs/miRs) miR156/529 [ 1 ]. Non-vascular land plants also undergo growth-phase transition to the reproductive state, but knowledge regarding the controlling mechanisms is limited. Here, we investigate the reproductive transition in the liverwort Marchantia polymorpha , focusing on the roles of miR529c [ 2–4 ] and MpSPL2. First, we established mir529c -null mutants using CRISPR/Cas9. Even in the absence of far-red light-supplemented long-day condition, which is usually needed to induce reproductive development [ 5, 6 ], the mutant thalli developed sexual reproductive organs (gametangia) and produced gametes. Transgenic plants expressing a miR529-resistant Mp SPL2 transgene also showed a similar phenotype of reproductive transition in the absence of inductive far-red light signals. In these mutants and transgenic plants, the Mp SPL2 mRNA abundance was elevated. Mp spl2 ko mutant plants showed successful gamete development and fertilization, which suggests that Mp SPL2 is involved in, but not essential for, sexual reproduction in M. polymorpha. Furthermore, analysis of Mp spl2 ko mutant and its complemented lines suggests that Mp SPL2 may have a role in promotion of reproductive transition. These findings support the notion that the transition to reproductive development in liverworts is controlled by a system similar to that in angiosperms, and the miR156/529- SPL module has common significance in the control of the vegetative-to-reproductive transition during development in many land plants, including liverworts. • Loss of miR529c causes gamete production in the absence of environmental signals • Repression of Mp SPL2 expression by miR529c prevents the reproductive transition • Mp SPL2 is involved in, but not essential for, reproductive development • The miR156/529- SPL module controls the reproductive transition in land plants Tsuzuki et al. report that the loss of miR529c in the liverwort Marchantia polymorpha causes gametangium formation and gamete production without the far-red light signaling needed for the reproductive transition, because of de-repressed expression of Mp SPL2. The miR156/529- SPL module generally controls the reproductive transition in land plants. [ABSTRACT FROM AUTHOR]

Daylength is perceived as a seasonal cue to induce growth-phase transition at a proper time of a year. The core of the mechanism of daylength measurement in angiosperms lies in the circadian clock-controlled expression of regulators of growth-phase transition. However, the roles of the circadian clock in daylength measurement in basal land plants remain largely unknown. In this study, we investigated the contribution of circadian clock to daylength measurement in a basal land plant, the liverwort Marchantia polymorpha . In M. polymorpha , transition from vegetative to reproductive phase under long-day conditions results in differentiation of sexual branches called gametangiophores which harbor gametangia. First, we showed that a widely used wild-type accession Takaragaike-1 is an obligate long-day plant with a critical daylength of about 10 hours and requires multiple long days. Then, we compared the timing of gametangiophore formation between wild type and circadian clock mutants in long-day and short-day conditions. Mutations in two clock genes, Mp TIMING OF CAB EXPRESSION 1 and Mp PSEUDO-RESPONSE REGULATOR , had no significant effects on the timing of gametangiophore formation. In addition, when M. polymorpha plants were treated with a chemical which lengthens circadian period, there was no significant effect on the timing of gametangiophore formation, either. We next observed the timing of gametangiophore formation under various non-24-h light/dark cycles to examine the effect of phase alteration in circadian rhythms. The results suggest that daylength measurement in M. polymorpha is based on the relative amount of light and darkness within a cycle rather than the intrinsic rhythms generated by circadian clock. Our findings suggest that M. polymorpha has a daylength measurement system which is different from that of angiosperms centered on the circadian clock function., 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 Kanesaka, Inoue, Tomita, Yamaoka and Araki.)

The vegetative cell nucleus proceeds ahead of a pair of sperm cells located beneath the pollen tube tip during germination. The tip-localized vegetative nucleus had been considered to play a pivotal role in the control of directional pollen tube growth and double fertilization. However, we recently reported the female-targeting behavior of pollen tubes from mutant plants, of which the vegetative nucleus and sperm nuclei were artificially immotile. We showed that the apical region of the mutant pollen tubes became physiologically enucleated after the first callose plug formation, indicating the autonomously growing nature of pollen tubes without the vegetative nucleus and sperm cells. Thus, in this study, we further analyzed another Arabidopsis thaliana mutant producing physiologically enucleated pollen tubes and discussed the mechanism by which a pollen tube can grow without de novo transcription from the vegetative nucleus. We propose several possible molecular mechanisms for persistent pollen tube growth, such as the contribution of transcripts before and immediately after germination and the use of persistent transcripts, which may be important for a competitive race among pollen tubes., 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 © 2022 Motomura, Sugi, Takeda, Yamaoka and Maruyama.)