Targeted single-cell gene induction by optimizing the dually regulated CRE/loxP system by a newly defined heat-shock promoter and the steroid hormone in Arabidopsis thaliana

Multicellular organisms rely on intercellular communication systems to organize their cellular functions. In studies focusing on intercellular communication, the key experimental techniques include the generation of chimeric tissue using transgenic DNA recombination systems represented by the CRE/loxP system. If an experimental system enables the induction of chimeras at highly targeted cell(s), it will facilitate the reproducibility and precision of experiments. However, multiple technical limitations have made this challenging. The stochastic nature of DNA recombination events, especially, hampers reproducible generation of intended chimeric patterns. Infrared laser-evoked gene operator (IR-LEGO), a microscopic system that irradiates targeted cells using an IR laser, can induce heat shock-mediated expression of transgenes, for example, CRE recombinase gene, in the cells. In this study, we developed a method that induces CRE/loxP recombination in the target cell(s) of plant roots and leaves in a highly specific manner. We combined IR-LEGO, an improved heat-shock-specific promoter, and dexamethasone-dependent regulation of CRE. The optimal IR-laser power and irradiation duration were estimated via exhaustive irradiation trials and subsequent statistical modeling. Under optimized conditions, CRE/loxP recombination was efficiently induced without cellular damage. We also found that the induction efficiency varied among tissue types and cellular sizes. The developed method offers an experimental system to generate a precisely designed chimeric tissue, and thus, will be useful for analyzing intercellular communication at high resolution in roots and leaves

Redox feedback regulation of ANAC089 signaling alters seed germination and stress response

21 p.-4 fig.-2 tab. 1 graph. abst.The interplay between the phytohormone abscisic acid (ABA) and the gasotransmitter nitric oxide (NO) regulates seed germination and post-germinative seedling growth. We show that GAP1 (germination in ABA and cPTIO 1) encodes the transcription factor ANAC089 with a critical membrane-bound domain and extranuclear localization. ANAC089 mutants lacking the membrane-tethered domain display insensitivity to ABA,salt, and osmotic and cold stresses, revealing a repressor function. Whole-genome transcriptional profiling and DNA-binding specificity reveals that ANAC089 regulates ABA- and redox-related genes. ANAC089 truncated mutants exhibit higher NO and lower ROS and ABA endogenous levels, alongside an altered thiol and disulfide homeostasis. Consistently, translocation of ANAC089 to the nucleus is directed by changes in cellular redox status after treatments with NO scavengers and redox-related compounds. Our results reveal ANAC089 to be a master regulator modulating redox homeostasis and NO levels, able to repress ABA synthesis and signaling during Arabidopsis seed germination and abiotic stress.Wethank the Spanish networks BIO2015-68957-REDT and RED2018-102397-T for stimulating discussions, as well as Dr. José M. Carrasco and Dr. Pablo Vera (IBMCP-CSIC) for help with the protein-expression experiments of the PBM. This work was financed by grants EcoSeed Impacts of Environmental Conditions on Seed Quality ‘‘EcoSeed-311840’’ ERC.KBBE.2012.1.1-01;BIO2017-85758-R and CSD2007-00057 (TRANSPLANTA) from the Ministerio de Ciencia, Innovación y Universidades (MICIU) (Spain); SA313P18 and SA137P20 from Junta de Castilla y León; Escalera de Excelencia CLU-2018-04 co-funded by the P.O. FEDER of Castilla y León 2014–2020 Spain (to O.L.); and the PhD and University Teacher Training Fellowship, Spanish Ministry of Science and Education (to P.A.).Peer reviewe

ニホン シホン シュギ カクリツキ ノ ミツビシ ザイバツ

論文タイプ||論

Identification of cis-Elements That Regulate Gene Expression during Initiation of Axillary Bud Outgrowth in Arabidopsis

Growth regulation associated with dormancy is an essential element in plant life cycles. To reveal regulatory mechanisms of bud outgrowth, we analyzed transcriptomes of axillary shoots before and after main stem decapitation in Arabidopsis (Arabidopsis thaliana). We searched for any enriched motifs among the upstream regions of up-regulated and down-regulated genes after decapitation. The promoters of down-regulated genes were enriched for TTATCC motifs that resemble the sugar-repressive element, whereas the promoters of up-regulated genes were enriched for GGCCCAWW and AAACCCTA, designated Up1 and Up2, respectively. Transgenic plants harboring a reporter gene driven by a tandem repeat of the elements were produced to analyze their function in vivo. Sugar-repressive element-mediated gene expression was down-regulated by the application of sugars but was unaffected after decapitation. In contrast, expression driven by the repeat containing both Up1 and Up2 was up-regulated after decapitation, although the Up1 or Up2 repeat alone failed to induce reporter gene expression in axillary shoots. In addition, disruption of both Up1 and Up2 elements in a ribosomal protein gene abolished the decapitation-induced expression. Ontological analysis demonstrated that up-regulated genes with Up elements were disproportionately predicted to function in protein synthesis and cell cycle. Up1 is similar to an element known to be a potential target for TCP (TEOSINTE BRANCHED1, CYCLOIDEA, PCFs family) transcription factor(s), which regulate expression of cell cycle-related and ribosomal protein genes. Our data indicate that Up1-mediated transcription of protein synthesis and cell cycle genes is an important regulatory step during the initiation of axillary shoot outgrowth induced by decapitation