Sheng Zhong, Peng Zhao, Xiongbo Peng, Hong-Ju Li, Qiaohong Duan, Alice Y Cheung; From gametes to zygote: mechanistic advances and emerging possibilities in plan

Most pentatricopeptide repeat (PPR) proteins are involved in organelle post-transcriptional processes, including RNA editing. The PPR proteins include the PLS subfamily, containing characteristic triplets of P, L, and S motifs; however, their editing mechanisms and roles in developmental processes are not fully understood. In this study, we isolated the Arabidopsis thaliana Growing slowly 1 ( AtGRS1 ) gene and showed that it functions in RNA editing and plant development. Arabidopsis null mutants of grs1 exhibit slow growth and sterility. Further analysis showed that cell division activity was reduced dramatically in the roots of grs1 plants. We determined that GRS1 is a nuclear-encoded mitochondria-localized PPR protein, and is a member of the PLS subfamily. GRS1 is responsible for the RNA editing at four specific sites of four mitochondrial mRNAs: nad1-265 , nad4L-55 , nad6-103 , and rps4-377 . The first three of these mRNAs encode for the subunits of complex I of the electron transport chain in mitochondria. Thus, the activity of complex I is strongly reduced in grs1 . Changes in RPS4 editing in grs1 plants affect mitochondrial ribosome biogenesis. Expression of the alternative respiratory pathway and the abscisic acid response gene ABI5 were up-regulated in grs1 mutant plant s . Genetic analysis revealed that ABI5 is involved in the short root phenotype of grs1 . Taken together, our results indicate that AtGRS1 regulates plant development by controlling RNA editing in Arabidopsis.

Ribosomal proteins are involved in numerous essential cell activities in plants. However, the regulatory role in specific plant developmental processes has not yet been fully elucidated. Here we identified the new ribosomal protein L18aB, which is specifically involved in sexual reproduction and plays a critical role in male gametophyte development and embryo pattern formation. In rpl18aB mutant plants, the mature pollen grains can germinate normally, but their competitiveness for growing in the style is significantly reduced. More interestingly, RPL18aB is required in early embryogenesis. rpl18aB embryos displayed irregular cell division orientations in the early pro-embryo and arrested at the globular stage with possible, secondary pattern formation defects. Further investigations revealed that the polar transportation of auxin is disturbed in the rpl18aB mutant embryos, which may explain the observed failure in embryo pattern formation. The cell type-specific complementation of RPL18aB in rpl18aB was not able to recover the phenotype, indicating that RPL18aB may play an essential role in early cell fate determination. This work unravels a novel role in embryo development for a ribosomal protein and provides insight into regulatory mechanism of early embryogenesis.

Significance: In a mature female gametophyte of angiosperm, one egg cell and two synergid cells, referred to as the “egg apparatus,” originate from the same functional megaspore. How the egg cell and synergid cells differentiate into distinct cell types has drawn much attention but remains unclear. In this work, a live imaging system was established to visualize nuclear movement and cell specialization in female gametophytes. The final localization of the nuclei before cellularization was found to be critical for determining the cell fates of the egg apparatus. Artificial changes in the nuclear position could produce two functional egg cells. Furthermore, the plant hormone auxin was suggested as a positional cue for egg apparatus specialization.

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. Significance Flowering plants have evolved a unique double-fertilization process along with an actin filament (F-actin)-based gamete nuclear migration mechanism. However, how dynamic F-actin movement is controlled in the female gametophytic cells remains unclear. We identified that the movement of F-actin is promoted via an ARP2/3-independent WAVE/SCAR-signaling pathway. We also discovered that the plant class XI myosin XI-G has a function involved in the active movement of F-actin required for sperm nuclear migration, which is different from the canonical myosin function as a cargo transporter. These breakthroughs also provide us with opportunities to further understand how flowering plants control double fertilization and plant cytoskeleton dynamics.