Abscisic acid root-to-shoot translocation by transporter AtABCG25 mediates stomatal movements in Arabidopsis.

The phytohormone abscisic acid (ABA) plays a central role in regulating stomatal movements under drought conditions. The root-derived peptide CLAVATA3/EMBRYO SURROUNDING REGION-RELATED 25 (CLE25) moves from the root to shoot for activating ABA biosynthesis under drought conditions. However, the root-to-shoot translocation of root-derived ABA and its regulation of stomatal movements in the shoot remain to be clarified. Here, we reveal that the ABA transporter ATP-binding cassette subfamily G member 25 (AtABCG25) mediates root-to-shoot translocation of ABA and ABA-glucosyl ester (ABA-GE) in Arabidopsis (Arabidopsis thaliana). Isotope-labeled ABA tracer experiments and hormone quantification in xylem sap showed that the root-to-shoot translocation of ABA and ABA-GE was substantially impaired in the atabcg25 mutant under nondrought and drought conditions. However, the contents of ABA and ABA-GE in the leaves were lower in the atabcg25 mutant than in the wild type (WT) under nondrought but similar under drought conditions. Consistently, the stomatal closure was suppressed in the atabcg25 mutant under nondrought but not under drought conditions. The transporter activity assays showed that AtABCG25 directly exported ABA and ABA-GE in planta and in yeast (Saccharomyces cerevisiae) cells. Thus, we proposed a working model in which root-derived ABA transported by AtABCG25 via xylem mediates stomatal movements in the shoot under nondrought conditions but might exhibit little effect on stomatal movements under drought conditions. These findings extend the functions of AtABCG25 and provide insights into the long-distance translocation of ABA and its role in stomatal movements.

Phloem unloading via the apoplastic pathway is essential for shoot distribution of root-synthesized cytokinins.

Root-synthesized cytokinins are transported to the shoot and regulate the growth, development, and stress responses of aerial tissues. Previous studies have demonstrated that Arabidopsis (Arabidopsis thaliana) ATP binding cassette (ABC) transporter G family member 14 (AtABCG14) participates in xylem loading of root-synthesized cytokinins. However, the mechanism by which these root-derived cytokinins are distributed in the shoot remains unclear. Here, we revealed that AtABCG14-mediated phloem unloading through the apoplastic pathway is required for the appropriate shoot distribution of root-synthesized cytokinins in Arabidopsis. Wild-type rootstocks grafted to atabcg14 scions successfully restored trans-zeatin xylem loading. However, only low levels of root-synthesized cytokinins and induced shoot signaling were rescued. Reciprocal grafting and tissue-specific genetic complementation demonstrated that AtABCG14 disruption in the shoot considerably increased the retention of root-synthesized cytokinins in the phloem and substantially impaired their distribution in the leaf apoplast. The translocation of root-synthesized cytokinins from the xylem to the phloem and the subsequent unloading from the phloem is required for the shoot distribution and long-distance shootward transport of root-synthesized cytokinins. This study revealed a mechanism by which the phloem regulates systemic signaling of xylem-mediated transport of root-synthesized cytokinins from the root to the shoot.

Spatially distributed cytokinins: Metabolism, signaling, and transport.

Cytokinins are mobile phytohormones that regulate plant growth, development, and environmental adaptability. The major cytokinin species include isopentenyl adenine (iP), trans-zeatin (tZ), cis-zeatin (cZ), and dihydrozeatin (DZ). The spatial distributions of different cytokinin species in different organelles, cells, tissues, and organs are primarily shaped by biosynthesis via isopentenyltransferases (IPT), cytochrome P450 monooxygenase, and 5'-ribonucleotide phosphohydrolase and by conjugation or catabolism via glycosyltransferase or cytokinin oxidase/dehydrogenase. Cytokinins bind to histidine receptor kinases in the endoplasmic reticulum or plasma membrane and relay signals to response regulators in the nucleus via shuttle proteins known as histidine phosphotransfer proteins. The movements of cytokinins from sites of biosynthesis to sites of signal perception usually require long-distance, intercellular, and intracellular transport. In the past decade, ATP-binding cassette (ABC) transporters, purine permeases (PUP), AZA-GUANINE RESISTANT (AZG) transporters, equilibrative nucleoside transporters (ENT), and Sugars Will Eventually Be Exported transporters (SWEET) have been characterized as involved in cytokinin transport processes. This review begins by introducing the spatial distributions of various cytokinins and the subcellular localizations of the proteins involved in their metabolism and signaling. Highlights focus on an inventory of the characterized transporters involved in cytokinin compartmentalization, including long-distance, intercellular, and intracellular transport, and the regulation of the spatial distributions of cytokinins by environmental cues. Future directions for cytokinin research are also discussed.

PROTACs Targeting BRM (SMARCA2) Afford Selective In Vivo Degradation over BRG1 (SMARCA4) and Are Active in BRG1 Mutant Xenograft Tumor Models.

The identification of VHL-binding proteolysis targeting chimeras (PROTACs) that potently degrade the BRM protein (also known as SMARCA2) in SW1573 cell-based experiments is described. These molecules exhibit between 10- and 100-fold degradation selectivity for BRM over the closely related paralog protein BRG1 (SMARCA4). They also selectively impair the proliferation of the H1944 "BRG1-mutant" NSCLC cell line, which lacks functional BRG1 protein and is thus highly dependent on BRM for growth, relative to the wild-type Calu6 line. In vivo experiments performed with a subset of compounds identified PROTACs that potently and selectively degraded BRM in the Calu6 and/or the HCC2302 BRG1 mutant NSCLC xenograft models and also afforded antitumor efficacy in the latter system. Subsequent PK/PD analysis established a need to achieve strong BRM degradation (>95%) in order to trigger meaningful antitumor activity in vivo. Intratumor quantitation of mRNA associated with two genes whose transcription was controlled by BRM (PLAU and KRT80) also supported this conclusion.

A fluorescence-based high-throughput screening method for cytokinin translocation mutants.

BACKGROUND: Cytokinins are one kind of phytohormones essential for plant growth, development and stress responses. In the past half century, significant progresses have been made in the studies of cytokinin signal transduction and metobolic pathways, but the mechanism of cytokinin translocation is poorly understood. Arabidopsis (Arabidopsis thaliana) response regulator 5 (ARR5) is a type-A response factor in cytokinin signaling which is induced by cytokinins and has been used as a reporter gene for the endogenous cytokinins in Arabidopsis. Here, we report a fluorescence-based high-throughput method to screen cytokinin translocation mutants using an ethyl methyl sulfone (EMS) mutagenesis library generated with ARR5::eGFP transgenic plants. RESULTS: The seedlings with enhanced green fluorescent protein (GFP) signal in roots were screened in a luminescence imaging system (LIS) in large scale to obtain mutants with over-accumulated cytokinins in roots. The selected mutants were confirmed under a fluorescence microscopy and then performed phenotypic analysis. In this way, we obtained twelve mutants with elevated GFP signal in the roots and further found three of them displayed reduced GFP signal in the aerial tissues. Two of the mutants were characterized and proved to be the atabcg14 allelic mutants which are defective in the long-distance translocation of root-synthesized cytokinins. CONCLUSIONS: We provide a strategy for screening mutants defective in cytokinin translocation, distribution or signaling. The strategy can be adapted to establish a system for screening mutants defective in other hormone transporters or signaling components using a fluorescence reporter.