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.

Cytokinins regulate rice lamina joint development and leaf angle.

Leaf angle is determined by lamina joint inclination and is an important agronomic trait that determines plant architecture, photosynthetic efficiency, and crop yield. Cytokinins (CKs) are phytohormones involved in shaping rice (Oryza sativa L.) architecture, but their role in leaf angle remains unknown. Here, we report that CK accumulation mediated by rice CK OXIDASE/DEHYDROGENASE3 (OsCKX3) controls lamina joint development and negatively regulates leaf angle. Phenotypic analysis showed that rice osckx3 mutants had smaller leaf angles, while the overexpression lines (OsCKX3-OE) had larger leaf angles. Histological sections indicated that the leaf inclination changes in the osckx3 and OsCKX3-OE lines resulted from asymmetric proliferation of the cells and vascular bundles in the lamina joint. Reverse transcription quantitative PCR, promoter-fused ß-glucuronidase expression, and subcellular localization assays indicated that OsCKX3 was highly expressed in the lamina joint, and OsCKX3-GFP fusion protein localized to the endoplasmic reticulum. The enzyme assays using recombinant protein OsCKX3 revealed that OsCKX3 prefers trans-zeatin (tZ) and isopentenyladenine (iP). Consistently, tZ and iP levels increased in the osckx3 mutants but decreased in the OsCKX3 overexpression lines. Interestingly, agronomic trait analysis of the rice grown in the paddy field indicated that osckx3 displayed a smaller leaf angle and enhanced primary branch number, grain size, 1,000-grain weight, and flag leaf size. Collectively, our results revealed that enhancing CK levels in the lamina joint by disrupting OsCKX3 negatively regulates leaf angle, highlighting that the CK pathway can be engineered to reduce leaf angle in rice and possibly in other cereals.

Esterase-Activated Precipitating Strategy to Achieve Highly Specific Detection and Long-Term Imaging of Calcium Ions by Aggregation-Induced Phosphorescence Probe.

Spatial and temporal monitoring of bioactive targets such as calcium ions is vitally significant for their essential roles in physiological and biochemical functions. Herein, we proposed an esterase-activated precipitating strategy to achieve highly specific identification and long-term bioimaging of calcium ions via lighting up the calcium ions by precipitation using a water-soluble aggregation-induced phosphorescence (AIP) probe. The designed probe CaP2 has an AIP behavior and can be efficiently aggregated by calcium ions through the coupling coordination of carboxylic acid and cyanide groups, which enables it to light up Ca2+ by precipitating-triggered phosphorescence. Four hydrophilic groups of tetraethylene glycol were introduced to endow the resulting probe CaP3 with extraordinary water solubility as well as excellent cellular penetration. Only when the probe CaP3 penetrates inside the live cells the existing esterase in cells can activate the probe to be transformed active CaP2 probe selectively binding with calcium ion in the surroundings. The probe was used to further evaluate the imaging of intracellular calcium ions in model organisms. The excellent imaging performance of CaP3 in Arabidopsis thaliana seedling roots demonstrates that CaP3 has the excellent capability of monitoring calcium ions in live-cell imaging, and furthermore CaP3 exhibits much better photostability and thereby greater potential in long-term imaging. This work established a general esterase-activated precipitating strategy to achieve specific detection and bioimaging in situ triggered by esterase in live cells, and established a water-soluble aggregation-induced phosphorescence probe with high selectivity to achieve specific sensing and long-term imaging of calcium ions in live cells.

Antipermeability Strategy to Achieve Extremely High Specificity and Ultralong Imaging of Diverse Cell Membranes Based on Restriction-Induced Emission of AIEgens.

Long-term in situ cell membrane-targeted bioimaging is of great significance for studying specific biological processes and functions, but currently developed membrane probes are rarely simultaneously used to image the plasma membrane of animal and plant cells, and these probes lack sufficiently high long-term targeting ability. Herein, we proposed an antipermeability strategy to achieve highly specific and long-term imaging of plasma membranes of both human and plant cells using the steric hindrance effect and restriction-induced emission of AIE-active probes based on an updated membrane model. A certain degree of rigidity of plasma membrane containing a large ratio of rigid cholesterol molecules in the updated membrane model provides a promising opportunity to design antipermeable probes by introducing a rigid steric hindrance group in the probe. The designed antipermeable probes can anchor inside plasma membrane for a long term relying on the combination of the steric hindrance effect and the electrostatic and hydrophobic interactions between the probe and the membrane, as well as light up the membrane via the restriction-induced emission mechanism. The excellent performance in imaging completeness and specificity for both human cells and plant cells clearly shows that these designed probes possess outstanding antipermeability to achieve long-term specific imaging of membrane. These probes also show some advanced features such as ultrafast staining, wash-free merit, favorable biocompatibility, good photostability, and effective resistance to viscosity and pH alteration. This work also provides a valuable design principle for membrane probes of plant cells that the designed probes require a suitable molecular size favoring the penetration of small pores of cell walls.

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.

ABC transporter OsABCG18 controls the shootward transport of cytokinins and grain yield in rice.

Cytokinins are one of the most important phytohormones and play essential roles in multiple life processes in planta. Root-derived cytokinins are transported to the shoots via long-distance transport. The mechanisms of long-distance transport of root-derived cytokinins remain to be demonstrated. In this study, we report that OsABCG18, a half-size ATP-binding cassette transporter from rice (Oryza sativa L.), is essential for the long-distance transport of root-derived cytokinins. OsABCG18 encodes a plasma membrane protein and is primarily expressed in the vascular tissues of the root, stem, and leaf midribs. Cytokinin profiling, as well as [14C]trans-zeatin tracer, and xylem sap assays, demonstrated that the shootward transport of root-derived cytokinins was significantly suppressed in the osabcg18 mutants. Transport assays in tobacco (Nicotiana benthamiana) indicated that OsABCG18 exhibited efflux transport activities for various substrates of cytokinins. While the mutation reduced root-derived cytokinins in the shoot and grain yield, overexpression of OsABCG18 significantly increased cytokinins in the shoot and improved grain yield. The findings for OsABCG18 as a transporter for long-distance transport of cytokinin provide new insights into the cytokinin transport mechanism and a novel strategy to increase cytokinins in the shoot and promote grain yield.

Feedback Regulation of DYT1 by Interactions with Downstream bHLH Factors Promotes DYT1 Nuclear Localization and Anther Development.

Transcriptional regulation is one of the most important mechanisms controlling development and cellular functions in plants and animals. The Arabidopsis thaliana bHLH transcription factor (TF) DYSFUNCTIONL TAPETUM1 (DYT1) is required for normal male fertility and anther development and activates the expression of the bHLH010/bHLH089/bHLH091 genes. Here, we showed that DYT1 is localized to both the cytoplasm and nucleus at anther stage 5 but specifically to the nucleus at anther stage 6 and onward. The bHLH010/bHLH089/bHLH091 proteins have strong nuclear localization signals, interact with DYT1, and facilitate the nuclear localization of DYT1. We further found that the conserved C-terminal BIF domain of DYT1 is required for its dimerization, nuclear localization, transcriptional activation activity, and function in anther development. Interestingly, when the BIF domain of DYT1 was replaced with that of bHLH010, the DYT1(N)-bHLH010(BIF) chimeric protein shows nuclear-preferential localization at anther stage 5 but could not fully rescue the dyt1-3 phenotype, suggesting that the normal spatio-temporal subcellular localization of DYT1 is important for DYT1 function and/or that the BIF domains from different bHLH members might be functionally distinct. Our results support an important positive feedback regulatory mechanism whereby downstream TFs increase the function of an upstream TF by enhancing its nucleus localization through the BIF domain.

The DYT1-interacting proteins bHLH010, bHLH089 and bHLH091 are redundantly required for Arabidopsis anther development and transcriptome.

The anther is the male reproductive organ of flowering plants, and the Arabidopsis bHLH transcription factors encoded by DYSFUNCTIONAL TAPETUM1 (DYT1) and ABORTED MICROSPORE (AMS) are required for control of the complex transcriptional networks regulating anther development. Knowledge of the mechanisms by which the bHLH proteins affect this diverse gene expression is quite limited. We examine here three recently duplicated Arabidopsis bHLH genes, bHLH010, bHLH089 and bHLH091, using evolutionary, genetic, morphological and transcriptomic approaches, and uncover their redundant functions in anther development. These three genes are relatively highly expressed in the tapetum of the Arabidopsis anther; single mutants at each of the bHLH010, bHLH089 and bHLH091 loci are developmentally normal, but the various double and triple combinations progressively exhibit increasingly defective anther phenotypes (abnormal tapetum morphology, delayed callose degeneration, and aborted pollen development), indicating their redundant functions in male fertility. Further transcriptomic and molecular analyses suggest that these three proteins act slightly later than DYT1, and also form protein complexes with DYT1, subsequently affecting the correct expression of many DYT1 target genes in the anther development transcriptional network. This study demonstrated that bHLH010, bHLH089 and bHLH091 together are important for the normal transcriptome of the developing Arabidopsis anther, possibly by forming a feed-forward loop with DYT1.

Target-activated multicolor fluorescent dyes for 3D imaging of plasma membranes and tracking of apoptosis.

Real-time tracking of dynamic changes in the three-dimensional morphology of the cell plasma membrane is of great importance for a deeper understanding of physiological processes related to the cell plasma membrane. However, there is a lack of imaging dyes that can specifically be used for a long term labelling of plasma membranes, especially for plant cells. Here, we have used molecular engineering strategies to develop a series of target-activated multicolour fluorescent dyes that can be used for long-term and three-dimensional imaging of plant cell plasma membranes. By combining different electron acceptors and donors, four molecular backbones with different emission colours from green to NIR have been obtained. In the designed styrene-based dyes, referred to as the SD dyes, several functional groups were introduced into the backbones to achieve the properties of target-activated fluorescence, rapid and wash-free staining, high plasma membrane targeting ability and long-term imaging function. Using onion epidermal cells as a platform, these dye molecules can provide high-quality imaging of the plasma membrane for up to 6 hours, providing a powerful tool for long-term monitoring of plasma membrane-related biological events. Calcium-mediated apoptosis of plant cells has been tracked for the first time by monitoring the morphological changes of the plasma membrane in real time using SD dyes. These dyes also exhibit excellent 3D imaging performance of the plasma membrane and were further used to track in real time the 3D morphological changes of the plasma membrane during plasmolysis of plant cells, providing a powerful imaging tool for three-dimensional (3D) biology. This work provides a set of multi-colour dye tools for long-term and three-dimensional imaging of plant cell plasma membranes, and also provides molecular design principles for guiding the transmembrane transport of small molecules.

Molecular engineering of fluorescent dyes for long-term specific visualization of the plasma membrane based on alkyl-chain-regulated cell permeability.

Long-term visualization of changes in plasma membrane dynamics during important physiological processes can provide intuitive and reliable information in a 4D mode. However, molecular tools that can visualize plasma membranes over extended periods are lacking due to the absence of effective design rules that can specifically track plasma membrane fluorescent dye molecules over time. Using plant plasma membranes as a model, we systematically investigated the effects of different alkyl chain lengths of FMR dye molecules on their performance in imaging plasma membranes. Our findings indicate that alkyl chain length can effectively regulate the permeability of dye molecules across plasma membranes. The study confirms that introducing medium-length alkyl chains improves the ability of dye molecules to target and anchor to plasma membranes, allowing for long-term imaging of plasma membranes. This provides useful design rules for creating dye molecules that enable long-term visualization of plasma membranes. Using the amphiphilic amino-styryl-pyridine fluorescent skeleton, we discovered that the inclusion of short alkyl chains facilitated rapid crossing of the plasma membrane by the dye molecules, resulting in staining of the cell nucleus and indicating improved cell permeability. Conversely, the inclusion of long alkyl chains hindered the crossing of the cell wall by the dye molecules, preventing staining of the cell membrane and demonstrating membrane impermeability to plant cells. The FMR dyes with medium-length alkyl chains rapidly crossed the cell wall, uniformly stained the cell membrane, and anchored to it for a long period without being transmembrane. This allowed for visualization and tracking of the morphological dynamics of the cell plasma membrane during water loss in a 4D mode. This suggests that the introduction of medium-length alkyl chains into amphiphilic fluorescent dyes can transform them from membrane-permeable fluorescent dyes to membrane-staining fluorescent dyes suitable for long-term imaging of the plasma membrane. In addition, we have successfully converted a membrane-impermeable fluorescent dye molecule into a membrane-staining fluorescent dye by introducing medium-length alkyl chains into the molecule. This molecular engineering of dye molecules with alkyl chains to regulate cell permeability provides a simple and effective design rule for long-term visualization of the plasma membrane, and a convenient and feasible means of chemical modification for efficient transmembrane transport of small molecule drugs.

Charge-regulated fluorescent anchors enable high-fidelity tracking of plasma membrane dynamics during biological events.

Many biological processes generally require long-term visualization tools for time-scale dynamic changes of the plasma membrane, but there is still a lack of design rules for such imaging tools based on small-molecule fluorescent probes. Herein, we revealed the key regulatory roles of charge number and species of fluorescent dyes in the anchoring ability of the plasma membrane and found that the introduction of multi-charged units and appropriate charge species is often required for fluorescent dyes with strong plasma membrane anchoring ability by systematically investigating the structure-function relationship of cyanostyrylpyridium (CSP) dyes with different charge numbers and species and their imaging performance for the plasma membrane. The CSP-DBO dye constructed exhibits strong plasma membrane anchoring ability in staining the plasma membrane of cells, in addition to many other advantages such as excellent biocompatibility and general universality of cell types. Such a fluorescent anchor has been successfully used to monitor chemically induced plasma membrane damage and dynamically track various cellular biological events such as cell fusion and cytokinesis over a long period of time by continuously monitoring the dynamic morphological changes of the plasma membrane, providing a valuable precise visualization tool to study the physiological response to chemical stimuli and reveal the structural morphological changes and functions of the plasma membrane during these important biological events from a dynamic perspective. Furthermore, CSP-DBO exhibits excellent biocompatibility and imaging capability in vivo such as labelling the plasma membrane in vivo and monitoring the metabolic process of lipofuscin as an aging indicator.

Long-term spatiotemporal and highly specific imaging of the plasma membrane of diverse plant cells using a near-infrared AIE probe.

Fluorescent probes are valuable tools to visualize plasma membranes intuitively and clearly and their related physiological processes in a spatiotemporal manner. However, most existing probes have only realized the specific staining of the plasma membranes of animal/human cells within a very short time period, while almost no fluorescent probes have been developed for the long-term imaging of the plasma membranes of plant cells. Herein, we designed an AIE-active probe with NIR emission to achieve four-dimensional spatiotemporal imaging of the plasma membranes of plant cells based on a collaboration approach involving multiple strategies, demonstrated long-term real-time monitoring of morphological changes of plasma membranes for the first time, and further proved its wide applicability to plant cells of different types and diverse plant species. In the design concept, three effective strategies including the similarity and intermiscibility principle, antipermeability strategy and strong electrostatic interactions were combined to allow the probe to specifically target and anchor the plasma membrane for an ultralong amount of time on the premise of guaranteeing its sufficiently high aqueous solubility. The designed APMem-1 can quickly penetrate cell walls to specifically stain the plasma membranes of all plant cells in a very short time with advanced features (ultrafast staining, wash-free, and desirable biocompatibility) and the probe shows excellent plasma membrane specificity without staining other areas of the cell in comparison to commercial FM dyes. The longest imaging time of APMem-1 can be up to 10 h with comparable performance in both imaging contrast and imaging integrity. The validation experiments on different types of plant cells and diverse plants convincingly proved the universality of APMem-1. The development of plasma membrane probes with four-dimensional spatial and ultralong-term imaging ability provides a valuable tool to monitor the dynamic processes of plasma membrane-related events in an intuitive and real-time manner.

Generation of the salicylic acid deficient Arabidopsis via a synthetic salicylic acid hydroxylase expression cassette.

BACKGROUND: Salicylic acid (SA) is one of the plant hormones, which plays crucial roles in signaling transduction in plant growth, disease resistance, and leaf senescence. Arabidopsis (Arabidopsis thaliana) SA 3-hydroxylase (S3H) and 5-hydroxylase (S5H) are key enzymes which maintain SA homeostasis by catalyzing SA to 2,3-dihydroxybenzoic acid (DHBA) and 2,5-DHBA, respectively. RESULTS: SA deficient transgenic Arabidopsis lines were generated by introducing two binary vectors S5Hpro::EGFP-S3H and 35Spro::EGFP-S3H respectively, in which the expression of S3H is under the control of the S5H promoter or CaMV 35S promoter. Compared with the constitutive expression of S3H gene under the control of 35S promoter, the S3H gene under the native S5H promoter is activated by endogenous SA and results in a dynamic control of SA catabolism in a feedback mode. The SA accumulation, growth, leaf senescence, and pathogen resistance of the S5Hpro::GFP-S3H transgenic plants were investigated in parallel with NahG transgenic plants. The SA levels in the S5Hpro::EGFP-S3H transgenic plants were similar to or slightly lower than those of NahG transgenic Arabidopsis and resulted in SA deficient phenotypes. The low-SA trait of the S5Hpro::EGFP-S3H transgenic lines was inherited stably in the later generations. CONCLUSIONS: Compared with NahG transgenic lines producing by-product catechol, S5Hpro::EGFP-S3H transgenic lines reduce SA levels by converting SA to a native product 2,3-DHBA for catabolism. Together, we provide new SA-deficient germplasms for the investigations of SA signaling in plant development, leaf senescence, and disease resistance.

Preparation and characterization of a new hydrophilic C60 derivative (OH)16C60CHCOOH.

C60 and its derivatives are very important nanomaterials in biological and materials science. Unfortunately, due to the high hydrophobicity of the C60 cage, most of these materials are insoluble in water and their usages are quitely restricted. Here we reported the preparation and characterization of (OH)16C60CHCOOH, a new multihydroxylated methanofullerene carboxylic acid (MMFCA) derivative with high aqueous solubility. The elementary analysis, FT-IR and 1HNMR confirmed the structure of this material. Like other C60 and its derivatives, this new derivative also contains the free radical scavenging ability, measured by electron spin resonance (ESR) test. On the other hand, the MTT assay showed it had low cytotoxicity. Therefore, this new MMFCA derivative might be a potential vehicle for the introduction of hydrophobic C60 derivatives into water or a hydrophilic environment.

Analyses of functional conservation and divergence reveal requirement of bHLH010/089/091 for pollen development at elevated temperature in Arabidopsis.

The Arabidopsis bHLH010/089/091 (basic helix-loop-helix) genes are functionally redundant and are required for both anther development and normal expression of DYT1-activated anther-related genes. These three genes are conserved in Brassicaceae, suggesting that each of them is under selection pressure; however, little is known about the possible functional differences among these bHLH genes and between the bHLH and DYT1 genes. Here, we compared novel anther transcriptomic data sets from bHLH010/089/091 single and double mutants, with an anther transcriptomic data set from the wild type (WT) and a previously obtained anther transcriptomic data set from the bhlh010 bhlh089 bhlh091 triple mutant. The results revealed molecular phenotypes that support the functional redundancy and divergence of bHLH010, bHLH089, and bHLH091, as well as the functional overlap and difference between them and DYT1. DNA-binding analyses revealed that DYT1 and bHLH089 specifically recognize the TCATGTGC box to activate the expression of target genes, including ATA20, EXL4, and MEE48. In addition, among genes whose expression was affected in the bhlh010 bhlh089 double and bhlh010 bhlh089 bhlh091 triple mutants, genes that are involved in the stress response and cell signaling were enriched, which included 256 genes whose expression was preferentially induced by heat during early flower development. Moreover, the bhlh double mutants exhibited defective pollen development when the plants were grown under elevated temperature, suggesting that bHLH genes are important for anther gene expression under such conditions. These results are consistent with the observation that the heat-induced expression of several genes is less in the bhlh mutants than that in the WT. Therefore, our results provide important insights into the molecular mechanism underlying the activation of direct targets by DYT1-bHLH089 heterodimers and demonstrate the protective roles of bHLH010/089/091 in maintaining fertility upon heat stress.

Trypsin-gold nanoparticle conjugates: binding, enzymatic activity, and stability.

Nanomaterials may interact with biomolecules in various ways and change their bioactivities. Here, we report on how gold nanoparticles (AuNPs) affect a most important protease, trypsin. After simple mixing of the trypsin and AuNPs solution the average diameter is 20 nm under enzyme friendly conditions (pH 8.0); the bond between trypsin and AuNPs was detected by UV-vis spectroscopy. The further protease assay of trypsin, before and after mixing with the AuNPs solution, pointed out an improved performance in terms of enzyme activity and stability.

Environmentally relevant concentrations of arsenite induces developmental toxicity and oxidative responses in the early life stage of zebrafish.

Arsenic (As) present in water is a nonignorable environmental issue, even at low concentrations (≤150 µg L-1). To evaluate the toxic effect of low concentrations of As, zebrafish at early life stage were exposed to 0, 25, 50, 75, or 150 µg L-1 AsIII for 120 h. Our results indicated that low concentration of AsIII decreased zebrafish larvae's survival rate to 85%, 89% and 86% at 50, 75 and 150 µg L-1. Furthermore, low concentrations of AsIII exposure caused oxidative stress (elevated superoxide dismutase (SOD) activity and influenced the mRNA transcriptional levels of Cu/ZnSOD and MnSOD) and damage (increased malondialdehyde levels). Meanwhile, zebrafish larvae regulated the mRNA transcription of metallothionein and heat shock protein 70 to alleviate toxicity caused by AsIII. These results revealed lower concentrations (≤150 µg L-1) of AsIII had a detriment effect on the survival of fish at early life stage, moreover, oxidative stress caused by AsIII posed potential risk for the zebrafish. This study provides novel insight into low concentration AsIII-induced toxicity in zebrafish.