The Arabidopsis thaliana U-box E3 ligases PUB18/PUB19 and PUB22/PUB23 are negative regulators of drought stress responses. PUB18/PUB19 regulate the drought stress response in an abscisic acid (ABA)-dependent manner, whereas PUB22/PUB23 regulate this response in an ABA-independent manner. A major structural difference between PUB18/PUB19 and PUB22/PUB23 is the presence of the UND (U-box N-terminal domain). Here, we focused on elucidating the molecular mechanism that mediates the functional difference between PUB18 and PUB22 and found that the UND was critically involved in the negative regulation of ABA-mediated stomatal movements. Exo70B1, a subunit of the exocyst complex, was identified as a target of PUB18, whereas Exo70B2 was a substrate of PUB22. However, the ∆UND-PUB18 derivative failed to ubiquitinate Exo70B1, but ubiquitinated Exo70B2. By contrast, the UND-PUB22 chimeric protein ubiquitinated Exo70B1 instead of Exo70B2, suggesting that the ubiquitination specificities of PUB18 and PUB22 to Exo70B1 and Exo70B2, respectively, are dependent on the presence or absence of the UND motif. The ABA-insensitive phenotypes of the pub18 pub19 exo70b1 triple mutant were reminiscent of those of exo70b1 rather than pub18 pub19, indicating that Exo70B1 functions downstream of PUB18. Overall, our results suggest that the UND motif is crucial for the negative regulation of ABA-dependent stomatal movement and for determination of its ubiquitination specificity to Exo70B1.

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Light sheet fluorescence microscopy (LSFM) has become an indispensable tool in biomedical studies owing to its depth-sectioning capability and low photo-bleaching. The axial resolution in LSFM is determined mainly by the thickness of the illumination sheet, and a high numerical-aperture lens is thus preferred in the illumination to increase the axial resolution. However, a rapid divergence of the illumination beam limits the effective field-of-view (FoV), that provides high-resolution images. Several strategies have been demonstrated for FoV enhancement, which involve the use of Bessel or Airy beams, for example. However, the generation of these beams requires complicated optical setup or phase filters with continuous phase distributions, which are difficult to manufacture. In contrast, a binary phase filter (BPF) comprising concentric rings with 0 or π phases produces a response similar to its continuous original and is easy to realize. Here, we present a novel form of LSFM that integrates BPFs derived from two representative axi-symmetric aberrations, including phase axicon and spherical aberrations, to improve the imaging performance. We demonstrate that these BPFs significantly increase the FoV, and those derived from axicon generate self-reconstructing beams, which are highly desirable in imaging through scattering specimens. We validate its high-contrast imaging capability over extended FoV by presenting three-dimensional images of microspheres, imaginal disc of Drosophila larva, and Arabidopsis.

The mechanism regulating proteasomal activity under proteotoxic stress conditions remains unclear. Here, we showed that arsenite-induced proteotoxic stress resulted in upregulation of Arabidopsis homologous PUB22 and PUB23 U-box E3 ubiquitin ligases and that pub22pub23 double mutants displayed arsenite-insensitive seed germination and root growth phenotypes. PUB22/ PUB23 downregulated 26S proteasome activity by promoting the dissociation of the 19S regulatory particle from the holo-proteasome complex, resulting in intracellular accumulation of Ub G76V -GFP, an artificial substrate of the proteasome complex, and insoluble poly-ubiquitinated proteins. These results suggest that PUB22/PUB23 play a critical role in arsenite-induced proteotoxic stress response via negative regulation of 26S proteasome integrity.

The liverwort Marchantia polymorpha, as the earliest land plant, is an important model system for studies of molecular evolution. Liverworts, which undergo a haplo-diplontic life cycle, spend most of their life as haploid gametophyte bodies, called thalli. In addition, the thalli can produce vegetative propagules, termed gemmae, conferring asexual reproduction in liverworts. Thus, to obtain sufficient isogenic tissues for genetic manipulation, the thallus is a suitable material. Here, we found that 23-to 25-d-old fully expanded thalli can be used for the genetic transformation with the pre-cultivation process, setting up the mature thalli pre-cultivation method. Next, we elucidated that 10-to 12-d-old young thalli can be used for the tissue-culture-based transformation in the absence of the pre-cultivation step, establishing the young thalli direct co-culture method without significant changes of transformation efficiency compared with the mature thalli pre-culture method. Overall, we set up the protocol using various stages of thalli of M. polymorpha with stable transformation efficiency. Mainly, we focused on reducing the hands-on labor time for performing the genetic transformation by widening the available stage of thalli for transformation or excluding the regenerating process of the thallus plantlets. In this report, we describe the detailed procedures employed under experimental conditions.