NRPB3,the Third Largest Subunit Of RNA Polymerase Ⅱ, Is Essential For Stomatal Patterning And Differentiation In Arabidopsis

Stomata, which consist of paired guard cells, are produced on the epidermis of almost all aerial organs of land plants. Turgor-driven stomatal movement controls gas exchange between plants and the environment, regulating efficiency of photosynthesis and water use in planta. Stomata have played crucial roles in the colonization of land by plants. Stomatal development represents an ideal system for analyzing pattern formation, cell division, and cell differentiation. Primary signal networks underlying stomatal development have been previously uncovered, however, much less is known about how these signals are transmitted to RNA polymerase II(Pol II) to regulate stomatal development. In eukaryotes, Pol II plays a central role in the transcription of mRNA-coding genes, most snRNAs, and microRNAs. The Arabidopsis Pol II contains 12 subunits. Homozygous T-DNA insertion mutants of Pol II genes are lethal, manifesting the importance of Pol II in plant development. Although the basic function of Pol II in mRNA transcription is understood, little is known about its other potential functions in plant cell differentiation.In this study, a partial loss-of-function mutation of the third largest subunit of nuclear DNA-dependent RNA polymerase II(NRPB3) that exhibited an increased number of stomatal lineage cells and paired stomata was identified. NRPB3 was broadly expressed in stomatal lineage cells and the protein was mainly localized in the cytoplasm and nucleus. Dex-inducible RNAi and artificial microRNAi gene-silencing plants of NRPB3(GVG-NRPB3 RNAi and amiR-NRPB3) displayed stomatal developmental defects including paired stomata, meristemoid-like cell clusters expressing TMMpro::nucGFP, and caterpillar-like structures expressing MUTEpro::GFP, indicating that NRPB3 is required for limiting stomatal lineage cell divisions. Genetic analysis showed that nrpb3 mutants dramatically exaggerated the phenotypes of stomatal mutants, such as tmm, er erl1 erl2, erl1 erl2, er105 erl2, er105, epf2, sdd1, flp, fama, ice1, and mute, suggesting that NRPB3 interacts synergistically with these genes in regulating stomatal patterning and differentiation. Protein-protein interaction assays showed that NRPB3 directly interacted with two basic-helix-loop-helix(bHLH) transcription factors, FAMA and INDUCER OF CBF EXPRESSION1(ICE1), and the mutation in nrpb3-1 influenced their physical interactions. Moreover, in GVG-NRPB3 RNAi, amiR-NRPB3, and FAMApro::amiR-NRPB3 transgenic plants, caterpillar-like structures similar to those in fama and ice1 mutants expressed FAMApro::nucGFP, further confirming the molecular interactions of NRPB3 with FAMA and ICE1 genetically. These results suggested that NRPB3 serves as an acceptor for stomatal development signals from transcription factors, FAMA and ICE1. This process is similar to the mechanism that the third largest subunit of animal Pol II(RPB3) associates with the bHLH transcription factor myogenin to regulate muscle cell differentiation.This study not only builds a direct linkage between the key component of the general transcription machinery(Pol II) to a specific developmental context(stomatal development pathway), but also reinforces the idea that mechanisms needed for the differentiation of skeletal muscle cell in animals are also required for stomatal development in plants.