Autophagy-mediated degradation of integumentary tapetum is critical for embryo pattern formation.

Autophagy modulates the degradation and recycling of intracellular materials and contributes to male gametophyte development and male fertility in plants. However, whether autophagy participates in seed development remains largely unknown. Here, we demonstrate that autophagy is crucial for timely programmed cell death (PCD) in the integumentary tapetum, the counterpart of anther tapetum, influencing embryo pattern formation and seed viability. Inhibition of autophagy resulted in delayed PCD of the integumentary tapetum and defects in embryo patterning. Cell-type-specific restoration of autophagic activities revealed that the integumentary tapetum plays a non-autonomous role in embryo patterning. Furthermore, high-throughput, comprehensive lipidomic analyzes uncovered an unexpected seed-developmental-stage-dependent role of autophagy in seed lipid metabolism: it contributes to triacylglycerol degradation before fertilization and to triacylglycerol biosynthesis after fertilization. This study highlights the critical role of autophagy in regulating timely integumentary tapetum PCD and reveals its significance in seed lipid metabolism and viability.

EGG CELL 1 contributes to egg-cell-dependent preferential fertilization in Arabidopsis.

During double fertilization in angiosperms, the pollen tube delivers two sperm cells into an embryo sac; one sperm cell fuses with an egg cell, and the other sperm cell fuses with the central cell. It has long been proposed that the preference for fusion with one or another female gamete cell depends on the sperm cells and occurs during gamete recognition. However, up to now, sperm-dependent preferential fertilization has not been demonstrated, and results on preferred fusion with either female gamete have remained conflicting. To investigate this topic, we generated Arabidopsis thaliana mutants that produce single sperm-like cells or whose egg cells are eliminated; we found that although the three different types of sperm-like cell are functionally equivalent in their ability to fertilize the egg and the central cell, each type of sperm-like cell fuses predominantly with the egg cell. This indicates that it is the egg cell that controls its preferential fertilization. We also found that sperm-activating small secreted EGG CELL 1 proteins are involved in the regulation of egg-cell-dependent preferential fertilization, revealing another important role for this protein family during double fertilization.

A female in vivo haploid-induction system via mutagenesis of egg cell-specific peptidases.

Crop breeding schemes can be significantly accelerated by using (doubled) haploid plants. In vivo haploid induction has been applied in plant breeding for decades but is still not available for all crops and genotypes, and haploidization rates are generally very low. Therefore, methodological improvements to and new concepts for haploidization are required. Here, we report a novel system for the induction of haploid plants by mutating genes encoding egg cell-specific aspartic endopeptidases (ECSs). We show that after successful sperm-egg cell fusion, ECSs play a critical role to ensure male and female nucleus fusion after fertilization. The ecs1 ecs2 double mutant can induce haploids by both selfing and hybridization in Arabidopsis and ECS mutation is also capable of producing haploids in rice. In summary, our study develops a novel approach for maternal haploidization and provides new insights into the molecular basis of fertilization.

The female germ unit is essential for pollen tube funicular guidance in Arabidopsis thaliana.

In angiosperm, two immotile sperm cells are delivered to the female gametes for fertilization by a pollen tube, which perceives guidance cues from ovules at least at two critical sites, micropyle for short-distance guidance and funiculus for comparably longer distance guidance. Compared with the great progress in understanding pollen tube micropylar guidance, little is known about the signaling for funicular guidance. Here, we show that funiculus plays an important role in pollen tube guidance and report that female gametophyte (FG) plays a critical role in funicular guidance by analysis of a 3-dehydroquinate synthase (DHQS) mutant. Loss function of DHQS in FG interrupts pollen tube funicular guidance, suggesting that the guiding signal is generated from FG. We show the evidence that the capacity of funicular guidance is established during FG functional specification after the establishment of cell identity. Specific expression of DHQS in the synergid cells, central cells, or egg cells can rescue funicular guidance defect in dhqs/+, indicating all the female germ unit cells are involved in the funicular guidance. The finding reveals that the attracting signal of pollen tube funicular guidance was generated at a site and stage manner and provides novel clue to locate and search for the signal.

DMP8 and 9 regulate HAP2/GCS1 trafficking for the timely acquisition of sperm fusion competence.

Sexual reproduction involves the fusion of two gametes of opposite sex. Although the sperm-expressed fusogen HAPLESS 2 (HAP2) or GENERATIVE CELL SPECIFIC 1 (GCS1) plays a vital role in this process in many eukaryotic organisms and an understanding of its regulation is emerging in unicellular systems [J. Zhang et al., Nat. Commun. 12, 4380 (2021); J. F. Pinello et al. Dev. Cell 56, 3380-3392.e9 (2021)], neither HAP2/GCS1 interactors nor mechanisms for delivery and activation at the fusion site are known in multicellular plants. Here, we show that Arabidopsis thaliana HAP2/GCS1 interacts with two sperm DUF679 membrane proteins (DMP8 and DMP9), which are required for the EGG CELL 1 (EC1)-induced translocation of HAP2/GCS1 from internal storage vesicle to the sperm plasma membrane to ensure successful fertilization. Our studies in Arabidopsis and tobacco provide evidence for a conserved function of DMP8/9-like proteins as HAP2/GCS1 partner in seed plants. Our data suggest that seed plants evolved a DMP8/9-dependent fusogen translocation process to achieve timely acquisition of sperm fusion competence in response to egg cell-derived signals, revealing a previously unknown critical step for successful fertilization.

MiR-1249 on Endothelial Extracellular Vesicles Mediates Cigarette Smoke-Induced Pulmonary Hypertension by Inhibiting HDAC10 (Histone Deacetylase 10)-NFκB (Nuclear Factor κB)-CaSR (Calcium-Sensing Receptor) Cascade.

BACKGROUND: Overproduction of endothelial extracellular vesicles (eEVs) is correlated with pulmonary hypertension progression, but the precise mechanism remains largely unclear. METHODS: MicroRNA-chip and real-time polymerase chain reaction were conducted to screen and validate microRNA profiles in blood plasma eEVs of rats and human with or without cigarette smoking. Pulmonary artery smooth muscle cells were cultured to study signaling pathways. Pulmonary hypertension phenotypes were evaluated in wild-type and calcium-sensing receptor knockout rats to identify the pathophysiological significance of the microRNA pathway. RESULTS: MicroR-1249 was predominant highly expressed in eEVs from plasma of rats exposed to cigarette smoking, and confirmed in eEVs from plasma of human smokers as well as in eEVs from cigarette smoke extract-treated pulmonary artery endothelial cells, but not in cigarette smoke extract-treated pulmonary artery smooth muscle cells. In cultured pulmonary artery smooth muscle cells, microR-1249 downregulated the expression of histone deacetylase 10, which in turn enhanced the acetylated form of NFκB (nuclear factor κB) level and its nuclear translocation leading to increased expression of calcium-sensing receptor. In rats, the repression of microR-1249 in eEVs by microR-1249 inhibitor, histone deacetylase 10 overexpression, or calcium-sensing receptor knockout profoundly inhibited the proliferative capacities and diminished apoptosis-resistance of pulmonary artery smooth muscle cells and pulmonary hypertension development in rats intravenously administrated with eEVs preparation from cigarette smoke extract-treated pulmonary artery endothelial cells. CONCLUSIONS: Cigarette smoke-enriched microR-1249 in endothelial extracellular vesicles facilitates the hyperproliferative and antiapoptotic status of pulmonary artery smooth muscle cells promoting pulmonary hypertension evolution through the inhibition of histone deacetylase 10-NFκB-calcium-sensing receptor cascade.

H3K27 methylation regulates the fate of two cell lineages in male gametophytes.

During angiosperm male gametogenesis, microspores divide to produce a vegetative cell (VC) and a male germline (MG), each with distinct cell fates. The mechanism underlying determination of the MG cell/VC fate remains an important area of research, with many unanswered questions. Here, we report that H3K27me3 is essential for VC fate commitment in male Arabidopsis thaliana gametophytes; H3K27me3 erasure contributes to MG cell fate initiation. VC-targeted H3K27me3 erasure disturbed VC development and shifted the VC fate toward a gamete destination, which suggests that MG cells require H3K27me3 erasure to trigger gamete cell fate. Multi-omics and cytological analyses confirmed the occurrence of extensive cell identity transition due to H3K27me3 erasure. Therefore, we experimentally confirmed that MG cell/VC fate is epigenetically regulated. H3K27 methylation plays a critical role in guiding MG cell/VC fate determination for pollen fertility in Arabidopsis. Our work also provides evidence for two previous hypotheses: the germline cell fate is specified by the differential distribution of unknown determinants and VC maintains the default microspore program (i.e. the H3K27me3 setting) while MG requires reprogramming.

SYP72 interacts with the mechanosensitive channel MSL8 to protect pollen from hypoosmotic shock during hydration.

In flowering plants, hydration of desiccated pollen grains on stigma is a prerequisite for pollen germination, during which pollen increase markedly in volume through water uptake, requiring them to survive hypoosmotic shock to maintain cellular integrity. However, the mechanisms behind the adaptation of pollen to this hypoosmotic challenge are largely unknown. Here, we identify the Qc-SNARE protein SYP72, which is specifically expressed in male gametophytes, as a critical regulator of pollen survival upon hypoosmotic shock during hydration. SYP72 interacts with the MSCS-LIKE 8 (MSL8) and is required for its localization to the plasma membrane. Intraspecies and interspecies genetic complementation experiments reveal that SYP72 paralogs and orthologs from green algae to angiosperms display conserved molecular functions and rescue the defects of Arabidopsis syp72 mutant pollen facing hypoosmotic shock following hydration. Our findings demonstrate a critical role for SYP72 in pollen resistance to hypoosmotic shock through the MSL8 cascade during pollen hydration.

New insights into cell-cell communications during seed development in flowering plants.

The evolution of seeds is a major reason why flowering plants are a dominant life form on Earth. The developing seed is composed of two fertilization products, the embryo and endosperm, which are surrounded by a maternally derived seed coat. Accumulating evidence indicates that efficient communication among all three seed components is required to ensure coordinated seed development. Cell communication within plant seeds has drawn much attention in recent years. In this study, we review current knowledge of cross-talk among the endosperm, embryo, and seed coat during seed development, and highlight recent advances in this field.

The parental contributions to early plant embryogenesis and the concept of maternal-to-zygotic transition in plants.

The maternal-to-zygotic transition (MZT) is a major developmental transition in the life cycles of animals. It consists of two associated processes: maternal transcript clearance and zygotic genome activation (ZGA). The concept of MZT has been controversially discussed in plants. In this short review, we summarize recent advances in understanding the timing of ZGA and the similarities and differences between ZGA in eudicots and monocots. We discuss the parental contributions to the transcriptome of the proembryo and parental control of early embryogenesis, and we examine distinct differences in the ZGA between animals and plants, update relevant concepts on MZT, and highlight outstanding questions in this field.

Autophagy in sexual plant reproduction: new insights.

Autophagy is a mechanism by which damaged or unwanted cells are degraded and their constituents recycled. Over the past decades, research focused on autophagy has expanded from yeast to mammals and plants, and the core machinery regulating autophagy appears to be conserved. In plants, autophagy has essential roles in responses to stressful conditions and also contributes to normal development, especially in the context of reproduction. Here, based on recent efforts to understand the roles and molecular mechanisms underlying autophagy, we highlight the specific roles of autophagy in plant reproduction and provide new insights for further studies.

H2O2 response gene 1/2 are novel sensors or responders of H2O2 and involve in maintaining embryonic root meristem activity in Arabidopsis thaliana.

Signal molecule hydrogen peroxide (H2O2) plays critical roles in various processes of plant development. However, H2O2 signaling network, especially the responders that sense and respond to the H2O2 signal remain largely unknown. Here we report two homologous genes H2O2 Response Gene 1 and 2 (HRG1/2) in Arabidopsis that could quickly respond to exogenous or endogenous H2O2. Knockdown of HRG1/2 facilitated seed germination while overexpression of HRG1/2 greatly retarded seed germination. ROS level in HRG1 overexpression roots was significantly lower than that in HRG1/2 mutants after H2O2 treatment. The expression level of enzymatic antioxidant DHAR3 was upregulated in HRG1 overexpression plants, suggesting that DHAR3 is downstream of HRG1. That the root meristem length and cell number were significantly reduced in hrg1-1 and hrg2-1 plants upon H2O2 treatment compared to that of HRG1 overexpression plants also approves the idea that HRGs function in H2O2 removal. Further evolutionary analysis indicates that this is a dicotyledon-specific pathway responsive to H2O2. Together, this work reveals HRG1/2 as novel H2O2 responders involved in ROS scavenging to ensure embryonic root meristem activity. These findings provide valuable clues for the of H2O2 signaling and root meristem regulation.

Fertilized egg cells secrete endopeptidases to avoid polytubey.

Upon gamete fusion, animal egg cells secrete proteases from cortical granules to establish a fertilization envelope as a block to polyspermy1-4. Fertilization in flowering plants is more complex and involves the delivery of two non-motile sperm cells by pollen tubes5,6. Simultaneous penetration of ovules by multiple pollen tubes (polytubey) is usually avoided, thus indirectly preventing polyspermy7,8. How plant egg cells regulate the rejection of extra tubes after successful fertilization is not known. Here we report that the aspartic endopeptidases ECS1 and ECS2 are secreted to the extracellular space from a cortical network located at the apical domain of the Arabidopsis egg cell. This reaction is triggered only after successful fertilization. ECS1 and ECS2 are exclusively expressed in the egg cell and transcripts are degraded immediately after gamete fusion. ECS1 and ESC2 specifically cleave the pollen tube attractor LURE1. As a consequence, polytubey is frequent in ecs1 ecs2 double mutants. Ectopic secretion of these endopeptidases from synergid cells led to a decrease in the levels of LURE1 and reduced the rate of pollen tube attraction. Together, these findings demonstrate that plant egg cells sense successful fertilization and elucidate a mechanism as to how a relatively fast post-fertilization block to polytubey is established by fertilization-induced degradation of attraction factors.

Endosperm development is an autonomously programmed process independent of embryogenesis.

The seeds of flowering plants contain three genetically distinct structures: the embryo, endosperm, and seed coat. The embryo and endosperm need to interact and exchange signals to ensure coordinated growth. Accumulating evidence has confirmed that embryo growth is supported by the nourishing endosperm and regulated by signals originating from the endosperm. Available data also support that endosperm development requires communication with the embryo. Here, using single-fertilization mutants, Arabidopsis thaliana dmp8 dmp9 and gex2, we demonstrate that in the absence of a zygote and embryo, endosperm initiation, syncytium formation, free nuclear cellularization, and endosperm degeneration occur as in the wild type in terms of the cytological process and time course. Although rapid embryo expansion accelerates endosperm breakdown, our findings strongly suggest that endosperm development is an autonomously organized process, independent of egg cell fertilization and embryo-endosperm communication. This work confirms both the altruistic and self-directed nature of the endosperm during coordinated embryo-endosperm development. Our findings provide insights into the intricate interaction between the two fertilization products and will help to distinguish the physiological roles of the signaling between endosperm and embryo. These findings also open new avenues in agro-biotechnology for crop improvement.

Dietary Geranylgeranyl Pyrophosphate Counteracts the Benefits of Statin Therapy in Experimental Pulmonary Hypertension.

BACKGROUND: The mevalonate pathway generates endogenous cholesterol and intermediates including geranylgeranyl pyrophosphate (GGPP). By reducing GGPP production, statins exert pleiotropic or cholesterol-independent effects. The potential regulation of GGPP homeostasis through dietary intake and the interaction with concomitant statin therapy is unknown. METHODS: We developed a sensitive high-pressure liquid chromatography technique to quantify dietary GGPP and conducted proteomics, qualitative real-time polymerase chain reaction screening, and Western blot to determine signaling cascades, gene expression, protein-protein interaction, and protein membrane trafficking in wild-type and transgenic rats. RESULTS: GGPP contents were highly variable depending on food source that differentially regulated blood GGPP levels in rats. Diets containing intermediate and high GGPP reduced or abolished the effects of statins in rats with hypoxia- and monocrotaline-induced pulmonary hypertension: this was rescuable by methyl-allylthiosulfinate and methyl-allylthiosulfinate-rich garlic extracts. In human pulmonary artery smooth muscle cells treated with statins, hypoxia activated RhoA in an extracellular GGPP-dependent manner. Hypoxia-induced ROCK2 (Rho associated coiled-coil containing protein kinase 2)/Rab10 (Ras-related protein rab-10) signaling was prevented by statin and recovered by exogenous GGPP. The hypoxia-activated RhoA/ROCK2 pathway in rat and human pulmonary artery smooth muscle cells upregulated the expression of Ca2+-sensing receptor (CaSR) and HIMF (hypoxia-induced mitogenic factor), a mechanism attenuated by statin treatment and regained with exogenous GGPP. Rab10 knockdown almost abrogated hypoxia-promoted CaSR membrane trafficking, a process diminished by statin and resumed by exogenous GGPP. Hypoxia-induced pulmonary hypertension was reduced in rats with CaSR mutated at the binding motif of HIMF and the interaction between dietary GGPP and statin efficiency was abolished. In humans fed a high GGPP diet, blood GGPP levels were increased. This abolished statin-lowering effects on plasma GGPP, and also on hypoxia-enhanced RhoA activity of blood monocytes that was rescued by garlic extracts. CONCLUSIONS: There is important dietary regulation of GGPP levels that interferes with the effects of statin therapy in experimental pulmonary hypertension. These observations rely on a key and central role of RhoA-ROCK2 cascade activation and Rab10-faciliated CaSR membrane trafficking with subsequent overexpression and binding of HIMF to CaSR. These findings warrant clinical investigation for the treatment of pulmonary hypertension and perhaps other diseases by combining statin with garlic-derived methyl-allylthiosulfinate or garlic extracts and thus circumventing dietary GGPP variations.

Epigenetic regulation and intercellular communication during male gametophyte development.

The male gametophyte of angiosperms has long been recognized as an ideal system for the study of the molecular mechanisms regulating cell fate determination. Recent findings on histone variants in two cell lineages, vegetative-cell-derived small interfering RNA and transposable element expression provide new power for relevant investigations.

Plant egg cell fate determination depends on its exact position in female gametophyte.

Plant fertilization involves both an egg cell, which fuses with a sperm cell, and synergid cells, which guide pollen tubes for sperm cell delivery. Therefore, egg and synergid cell functional specifications are prerequisites for successful fertilization. However, how the egg and synergid cells, referred to as the "egg apparatus," derived from one mother cell develop into distinct cell types remains an unanswered question. In this report, we show that the final position of the nuclei in female gametophyte determines the cell fate of the egg apparatus. We established a live imaging system to visualize the dynamics of nuclear positioning and cell identity establishment in the female gametophyte. We observed that free nuclei should migrate to a specific position before egg apparatus specialization. Artificial changing in the nuclear position on disturbance of the actin cytoskeleton, either in vitro or in vivo, could reset the cell fate of the egg apparatus. We also found that nuclei of the same origin moved to different positions and then showed different cell identities, whereas nuclei of different origins moved to the same position showed the same cell identity, indicating that the final positions of the nuclei, rather than specific nucleus lineage, play critical roles in the egg apparatus specification. Furthermore, the active auxin level was higher in the egg cell than in synergid cells. Auxin transport inhibitor could decrease the auxin level in egg cells and impair egg cell identity, suggesting that directional and accurate auxin distribution likely acts as a positional cue for egg apparatus specialization.

AtMIF1 increases seed oil content by attenuating GL2 inhibition.

Vegetable oil is a major edible oil and an important industrial raw material. However, breeders have found it challenging to improve the oil content of crop seeds, and little is known about regulators with the potential to increase oil content via molecular engineering in modern oil crop breeding. We reported an F-box protein, Arabidopsis thaliana MYB Interaction Factor 1 (AtMIF1), which is a member of the ubiquitin-protein ligase E3 complex involved in the 26S proteasome protein degradation pathway. AtMIF1 physically interacts with MYB domain protein 5 (MYB5), which results in MYB5 degradation, so that transcriptional activation of the MYB/bHLH/WD-repeat (MBW) complex does not occur normally and GLABRA2 (GL2), encoding an inhibitor of oil content and functioning as a direct downstream gene of MBW, is not properly transcribed. AtMIF1 functioned as a positive regulator that increases oil content by attenuating GL2 inhibition. We overexpressed AtMIF1 and obtained transgenic plants with significantly higher seed oil contents. Importantly, both vegetative and reproductive growth of the transgenic plants appeared normal. In summary, this work reveals a novel regulator, AtMIF1, and a new regulatory pathway, 26S proteasome-AtMIF1-MYB5, for increasing the oil content of seeds without affecting plant growth, thus facilitating oil crop breeding.

ARP2/3-independent WAVE/SCAR pathway and class XI myosin control sperm nuclear migration in flowering plants.

After eukaryotic fertilization, gamete nuclei migrate to fuse parental genomes in order to initiate development of the next generation. In most animals, microtubules control female and male pronuclear migration in the zygote. Flowering plants, on the other hand, have evolved actin filament (F-actin)-based sperm nuclear migration systems for karyogamy. Flowering plants have also evolved a unique double-fertilization process: two female gametophytic cells, the egg and central cells, are each fertilized by a sperm cell. The molecular and cellular mechanisms of how flowering plants utilize and control F-actin for double-fertilization events are largely unknown. Using confocal microscopy live-cell imaging with a combination of pharmacological and genetic approaches, we identified factors involved in F-actin dynamics and sperm nuclear migration in Arabidopsis thaliana (Arabidopsis) and Nicotiana tabacum (tobacco). We demonstrate that the F-actin regulator, SCAR2, but not the ARP2/3 protein complex, controls the coordinated active F-actin movement. These results imply that an ARP2/3-independent WAVE/SCAR-signaling pathway regulates F-actin dynamics in female gametophytic cells for fertilization. We also identify that the class XI myosin XI-G controls active F-actin movement in the Arabidopsis central cell. XI-G is not a simple transporter, moving cargos along F-actin, but can generate forces that control the dynamic movement of F-actin for fertilization. Our results provide insights into the mechanisms that control gamete nuclear migration and reveal regulatory pathways for dynamic F-actin movement in flowering plants.