Straightforward and affordable agroinfiltration with RUBY accelerates RNA silencing research.

Transient expression and induction of RNA silencing by agroinfiltration is a fundamental method in plant RNA biology. Here, we introduce a new reporter assay using RUBY, which encodes three key enzymes of the betalain biosynthesis pathway, as a polycistronic mRNA. The red pigmentation conferred by betalains allows visual confirmation of gene expression or silencing levels without tissue disruption, and the silencing levels can be quantitatively measured by absorbance in as little as a few minutes. Infiltration of RUBY in combination with p19, a well-known RNA silencing suppressor, induced a fivefold higher accumulation of betalains at 7 days post infiltration compared to infiltration of RUBY alone. We demonstrated that co-infiltration of RUBY with two RNA silencing inducers, targeting either CYP76AD1 or glycosyltransferase within the RUBY construct, effectively reduces RUBY mRNA and betalain levels, indicating successful RNA silencing. Therefore, compared to conventional reporter assays for RNA silencing, the RUBY-based assay provides a simple and rapid method for quantitative analysis without the need for specialized equipment, making it useful for a wide range of RNA silencing studies.

A non-canonical BZR/BES transcription factor regulates the development of haploid reproductive organs in Marchantia polymorpha.

Gametogenesis, which is essential to the sexual reproductive system, has drastically changed during plant evolution. Bryophytes, lycophytes and ferns develop reproductive organs called gametangia-antheridia and archegonia for sperm and egg production, respectively. However, the molecular mechanism of early gametangium development remains unclear. Here we identified a 'non-canonical' type of BZR/BES transcription factor, MpBZR3, as a regulator of gametangium development in a model bryophyte, Marchantia polymorpha. Interestingly, overexpression of MpBZR3 induced ectopic gametangia. Genetic analysis revealed that MpBZR3 promotes the early phase of antheridium development in male plants. By contrast, MpBZR3 is required for the late phase of archegonium development in female plants. We demonstrate that MpBZR3 is necessary for the successful development of both antheridia and archegonia but functions in a different manner between the two sexes. Together, the functional specialization of this 'non-canonical' type of BZR/BES member may have contributed to the evolution of reproductive systems.

Possible molecular mechanisms of persistent pollen tube growth without de novo transcription.

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.

Persistent directional growth capability in Arabidopsis thaliana pollen tubes after nuclear elimination from the apex.

During the double fertilization process, pollen tubes deliver two sperm cells to an ovule containing the female gametes. In the pollen tube, the vegetative nucleus and sperm cells move together to the apical region where the vegetative nucleus is thought to play a crucial role in controlling the direction and growth of the pollen tube. Here, we report the generation of pollen tubes in Arabidopsis thaliana whose vegetative nucleus and sperm cells are isolated and sealed by callose plugs in the basal region due to apical transport defects induced by mutations in the WPP domain-interacting tail-anchored proteins (WITs) and sperm cell-specific expression of a dominant mutant of the CALLOSE SYNTHASE 3 protein. Through pollen-tube guidance assays, we show that the physiologically anuclear mutant pollen tubes maintain the ability to grow and enter ovules. Our findings provide insight into the sperm cell delivery mechanism and illustrate the independence of the tip-localized vegetative nucleus from directional growth control of the pollen tube.

AtNOT1 Is a Novel Regulator of Gene Expression during Pollen Development.

Development of pollen, the male gametophyte of flowering plants, is tightly controlled by dynamic changes in gene expression. Recent research to clarify the molecular aspects of pollen development has revealed the involvement of several transcription factors in the induction of gene expression. However, limited information is available about the factors involved in the negative regulation of gene expression to eliminate unnecessary transcripts during pollen development. In this study, we revealed that AtNOT1 is an essential protein for proper pollen development and germination capacity. AtNOT1 is a scaffold protein of the AtCCR4-NOT complex, which includes multiple components related to mRNA turnover control in Arabidopsis. Phenotypic analysis using atnot1 heterozygote mutant pollen showed that the mature mutant pollen failed to germinate and also revealed abnormal localization of nuclei and a specific protein at the tricellular pollen stage. Furthermore, transcriptome analysis of atnot1 heterozygote mutant pollen showed that the downregulation of a large number of transcripts, along with the upregulation of specific transcripts required for pollen tube germination by AtNOT1 during late microgametogenesis, is important for proper pollen development and germination. Overall, our findings provide new insights into the negative regulation of gene expression during pollen development, by showing the severely defective phonotype of atnot1 heterozygote mutant pollen.

A pharmacological study of Arabidopsis cell fusion between the persistent synergid and endosperm.

Cell fusion is a pivotal process in fertilization and multinucleate cell formation. A plant cell is ubiquitously surrounded by a hard cell wall, and very few cell fusions have been observed except for gamete fusions. We recently reported that the fertilized central cell (the endosperm) absorbs the persistent synergid, a highly differentiated cell necessary for pollen tube attraction. The synergid-endosperm fusion (SE fusion) appears to eliminate the persistent synergid from fertilized ovule in Arabidopsis thaliana Here, we analyzed the effects of various inhibitors on SE fusion in an in vitro culture system. Different from other cell fusions, neither disruption of actin polymerization nor protein secretion impaired SE fusion. However, transcriptional and translational inhibitors decreased the SE fusion success rate and also inhibited endosperm division. Failures of SE fusion and endosperm nuclear proliferation were also induced by roscovitine, an inhibitor of cyclin-dependent kinases (CDK). These data indicate unique aspects of SE fusion such as independence of filamentous actin support and the importance of CDK-mediated mitotic control.

Interconnections between mRNA degradation and RDR-dependent siRNA production in mRNA turnover in plants.

Accumulation of an mRNA species is determined by the balance between the synthesis and the degradation of the mRNA. Individual mRNA molecules are selectively and actively degraded through RNA degradation pathways, which include 5'-3' mRNA degradation pathway, 3'-5' mRNA degradation pathway, and RNA-dependent RNA polymerase-mediated mRNA degradation pathway. Recent studies have revealed that these RNA degradation pathways compete with each other in mRNA turnover in plants and that plants have a hidden layer of non-coding small-interfering RNA production from a set of mRNAs. In this review, we summarize the current information about plant mRNA degradation pathways in mRNA turnover and discuss the potential roles of a novel class of the endogenous siRNAs derived from plant mRNAs.

Fertilization-independent Cell-fusion between the Synergid and Central Cell in the Polycomb Mutant.

In flowering plants, fertilization of the central cell gives rise to an embryo-nourishing endosperm. Recently, we reported that the endosperm absorbs the adjacent synergid cell through a cell-fusion, terminating the pollen tube guidance by a rapid inactivation of the synergid cell. Although this synergid-endosperm fusion (SE fusion) initiates soon after fertilization, it was still unknown whether the triggers of SE fusion are stimuli during fertilization or other seed developmental processes. To further dissect out the SE fusion process, we investigated the SE fusion in an Arabidopsis mutant defective for MULTICOPY SUPPRESSOR OF IRA1 (MSI1), a subunit of the polycomb repressive complex 2 (PRC2). The mutant msi1 develops autonomous endosperm without fertilization. Time-lapse imaging revealed a rapid efflux of the synergid contents during the autonomous endosperm development, indicating that the initiation of SE fusion is under the control of some of the events triggered by fertilization of the central cell distinct from the discharge of pollen tube contents and plasma membrane fusion.

Diffuse decapping enzyme DCP2 accumulates in DCP1 foci under heat stress in Arabidopsis thaliana.

The decapping enzymes DCP1 and DCP2 are components of a decapping complex that degrades mRNAs. DCP2 is the catalytic core and DCP1 is an auxiliary subunit. It has been assumed that DCP1 and DCP2 are consistently co-localized in cytoplasmic RNA granules called processing bodies (P-bodies). However, it has not been confirmed whether DCP1 and DCP2 co-localize in Arabidopsis thaliana. In this study, we generated DCP1-green fluorescent protein (GFP) and DCP2-GFP transgenic plants that complemented dcp1 and dcp2 mutants, respectively, to see whether localization of DCP2 is identical to that of DCP1. DCP2 was present throughout the cytoplasm, whereas DCP1 formed P-body-like foci. Use of DCP1-GFP/DCP2-red fluorescent protein (RFP) or DCP1-RFP/DCP2-GFP plants showed that heat treatment induced DCP2 assembly into DCP1 foci. In contrast, cold treatment did not induce DCP2 assembly, while the number of DCP1 foci increased. These changes in DCP1 and DCP2 localization during heat and cold treatments occurred without changes in DCP1 and DCP2 protein abundance. Our results show that DCP1 and DCP2 respond differently to environmental changes, indicating that P-bodies have diverse DCP1 and DCP2 proportions depending on environmental conditions. The localization changes of DCP1 and DCP2 may explain how specific mRNAs are degraded during changes in environmental conditions.

The role of decapping proteins in the miRNA accumulation in Arabidopsis thaliana.

Decapping 1 (DCP1), Decapping 2 (DCP2) and VARICOSE (VCS) are components of the decapping complex that removes the 7-methyl-guanosine 5'-diphosphate from the 5' end of mRNAs. In animals, the decapping proteins are involved in miRNA-mediated gene silencing, whereas in plants the roles of the decapping proteins in the miRNA pathway are not well understood. Here we demonstrated that the accumulation of miRNAs decreased in dcp1, dcp2 and vcs mutants, indicating that DCP1, DCP2 and VCS are important for the miRNA pathway in Arabidopsis thaliana. The primary miRNAs (pri-miRNAs) did not increase and miRNA biogenesis components did not decrease in these mutants, suggesting that the miRNA decrease in decapping mutants is not due to the defect of pri-miRNA processing. We showed that the accumulation of miRNA targets increased concomitantly with the decrease of miRNA in the decapping mutants. Our results suggested that the seedling lethal phenotypes in the dcp1, dcp2 and vcs mutants are caused not only by the defect in decapping, but also by the disruption of miRNA-mediated gene regulation.