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Plant roots are sustained through meristem activity at the root tip. Two transcriptional pathways, one mediated by PLETHORAs (PLTs) and the other by SHORTROOT and SCARECROW, play major roles in root meristem development. The role of PLTs during root meristem development requires a concentration gradient, which is not only contributed by posttranslational regulation such as growth dilution and intercellular movement but also likely by a largely unknown fine-tuned transcriptional regulatory mechanism. We report here that Arabidopsis (Arabidopsis thaliana) JANUS positively regulates PLT1 expression in the root meristem by recruiting RNA polymerase II (Pol II) to PLT1 and by interacting with PLT1. JANUS-dependent recruitment of Pol II is inhibited through the competitive binding of JANUS by GRF-INTERACTING FACTOR1 (GIF1)/ANGUSTIFOLIA3, a transcriptional cofactor that negatively regulates PLT1 expression. Finally, GIF1 and JANUS, the antagonistic regulators of PLT1, both depend on Arabidopsis IMPORTIN β4 for their nuclear accumulation. The combination of an importin and its two antagonistic cargos in PLT1 transcription may have logistic benefits in fine-tuning the transcription of PLT1 in root meristem.

Male and female gametophytes are generated from micro- or megaspore mother cells through consecutive meiotic and mitotic cell divisions. Defects in these divisions often result in gametophytic lethality. Gametophytic lethality was also reported when genes encoding ribosome-related proteins were mutated. Although numerous ribosomal proteins (RPs) have been identified in plants based on homology with their yeast and metazoan counterparts, how RPs are regulated, e.g., through dynamic subcellular targeting, is unknown. We report here that an Arabidopsis (Arabidopsis thaliana) importin β, KETCH1 (karyopherin enabling the transport of the cytoplasmic HYL1), is critical for gametogenesis. Karyopherins are molecular chaperones mediating nucleocytoplasmic protein transport. However, the role of KETCH1 during gametogenesis is independent of HYPONASTIC LEAVES 1 (HYL1), a previously reported KETCH1 cargo. Instead, KETCH1 interacts with several RPs and is critical for the nuclear accumulation of RPL27a, whose mutations caused similar gametophytic defects. We further showed that knocking down KETCH1 caused reduced ribosome biogenesis and translational capacity, which may trigger the arrest of mitotic cell cycle progression and lead to gametophytic lethality.

Summary Embryonic pattern formation relies on positional coordination of cell division and specification. Early axis formation during Arabidopsis embryogenesis requires WUSCHEL RELATED HOMEOBOX (WOX)-mediated transcription activation and PIN-FORMED7 (PIN7)-mediated auxin asymmetry. How these events are regulated is obscure. We report that Arabidopsis JANUS, a putative subunit of spliceosome, is essential for embryonic pattern formation. Significantly reduced transcription but not mRNA processing of WOX2 and PIN7 in janus suggested its role in transcriptional regulation. JANUS interacts with RNA polymerase II (Pol II) through a region outside of its spliceosome-association domain. We further show that Pol II mediates the transcription of WOX2 and PIN7 in a JANUS-dependent way and is essential for embryonic pattern formation. These findings reveal that JANUS recruits Pol II for the activation of two parallel pathways to ensure proper pattern formation during embryogenesis., Graphical Abstract, Highlights • Arabidopsis JANUS, a putative spliceosome subunit, is essential for embryogenesis • JANUS mediates the transcription but not RNA processing of WOX2 and PIN7 • JANUS interacts with RNA polymerase II whose mutations caused embryo lethality • Pol II mediates the transcription of WOX2 and PIN7 in a JANUS-dependent manner, Biological Sciences; Molecular Biology; Plant Biology; Plant Development

Ovule development is critical for seed development and plant reproduction. Multiple transcription factors (TFs) have been reported to mediate ovule development. However, it is not clear which intracellular components regulate these TFs during ovule development. After their synthesis, TFs are transported into the nucleus a process regulated by karyopherins commonly known as importin alpha and β. Around half of Arabidopsis (Arabidopsis thaliana) importin β-coding genes have been functionally characterized but only two with specific cargos have been identified. We report here that Arabidopsis IMPORTIN β4 (IMB4) regulates ovule development through nucleocytoplasmic transport of transcriptional coactivator growth regulating factors–interacting factors (GIFs). Mutations in IMB4 impaired ovule development by affecting integument growth. imb4 mutants were also defective in embryo sac development, leading to partial female sterility. IMB4 directly interacts with GIFs and is critical for the nucleocytoplasmic transport of GIF1. Finally, functional loss of GIFs resulted in ovule defects similar to those in imb4 mutants, whereas enhanced expression of GIF1 partially restored the fertility of imb4. The results presented here uncover a novel genetic pathway regulating ovule development and reveal the upstream regulator of GIFs.

Pollen development is a pre-requisite for sexual reproduction of angiosperms, during which various cellular activities are involved. Pollen development accompanies dynamic remodeling of vacuoles through fission and fusion, disruption of which often compromises pollen viability. We previously reported that the Y subunit of adaptor protein 1 (AP1G) mediates synergid degeneration during pollen tube reception. Here, we demonstrate that AP1G is essential for pollen development. AP1G loss-of-function resulted in male gametophytic lethality due to defective pollen development. By ultrastructural analysis and fluorescence labeling, we demonstrate that AP1G loss-of-function compromised dynamic vacuolar remodeling during pollen development and impaired vacuolar acidification of pollen. Results presented here support a key role of vacuoles in gametophytic pollen development.

Double fertilization in angiosperms requires the delivery of immotile sperm through pollen tubes, which enter embryo sacs to initiate synergid degeneration and to discharge. This fascinating process, called pollen tube reception, involves extensive communications between pollen tubes and synergids, within which few intracellular regulators involved have been revealed. Here, we report that vacuolar acidification in synergids mediated by AP1G and V-ATPases might be critical for pollen tube reception. Functional loss of AP1G or VHA-A, encoding the γ subunit of adaptor protein 1 or the shared component of two endomembrane V-ATPases, respectively, impaired synergid-controlled pollen tube reception and caused partial female sterility. AP1G works in parallel to the plasma membrane-associated receptor FERONIA in synergids, suggesting that synergid-mediated pollen tube reception requires proper sorting of vacuolar cargos by AP1G. Although AP1G did not mediate the targeting of V-ATPases, AP1G loss of function or the expression of AP1G-RNAi compromised vacuolar acidification mediated by V-ATPases, implying their genetic interaction. We propose that vacuolar acidification might represent a distinct cell-death mechanism specifically adopted by the plant phylum, which is critical for synergid degeneration during pollen tube reception.

Planar morphogenesis, a distinct feature of multicellular organisms, is crucial for the development of ovule, progenitor of seeds. Both receptor-like kinases (RLKs) such as STRUBBELIG (SUB) and auxin gradient mediated by PIN-FORMED1 (PIN1) play instructive roles in this process. Fine-tuned intercellular communications between different cell layers during ovule development demands dynamic membrane distribution of these cell-surface proteins, presumably through vesicle-mediated sorting. However, the way it’s achieved and the trafficking routes involved are obscure. We report that HAPLESS13 (HAP13)-mediated trafficking of SUB is critical for ovule development. HAP13 encodes the μ subunit of adaptor protein 1 (AP1) that mediates protein sorting at the trans-Golgi network/early endosome (TGN/EE). The HAP13 mutant, hap13-1, is defective in outer integument growth, resulting in exposed nucellus accompanied with impaired pollen tube guidance and reception. SUB is mis-targeted in hap13-1. However, unlike that of PIN2, the distribution of PIN1 is independent of HAP13. Genetic interference of exocytic trafficking at the TGN/EE by specifically downregulating HAP13 phenocopied the defects of hap13-1 in SUB targeting and ovule development, supporting a key role of sporophytically expressed SUB in instructing female gametogenesis., Author Summary Ovules, being the progenitors of seeds, hold an important role in crop production. Ovule is a complex organ connecting two generations, i.e. diploid sporophytic cells and haploid female gametophytes (FG). Fertilization of female gametes leads to the development of embryo and endosperm while the sporophytic cells of ovules become seed coats. Because of its importance for plant reproduction, the development of ovules is tightly controlled. The integument cells are divided in a polar way so that integument cell layers fully enclose a mature FG, leaving an opening only for pollen tube entrance. The integuments also instruct FG development, synchronizing the two generations. Receptor-like kinases, especially STRUBBELIG (SUB), are crucial both for the establishment of ovular polarity and the intercellular communication between sporophytic and gametophytic cells. However, how SUB is post-translationally regulated, as would be expected for cell-surface proteins, is unclear. We report here that the dynamic trafficking of SUB via a subpopulation of endosomes is crucial for its functionality during ovule development. The membrane association of SUB was impaired when the recycling route from the trans-Golgi network/early endosome to the plasma membrane was disrupted in hap13-1, a mutant of HAPLESS13. We further demonstrate that defects of SUB targeting in the outer integuments compromised FG development, suggesting an intriguing intercellular communication. Our results highlight the importance of vesicular trafficking in the establishment of ovular polarity and in the communications between spatially separated cells.