A novel Escherichia coli cell-based bioreporter for quantification of salicylic acid in cosmetics.

Transcription factor-based bioreporters have been extensively studied for monitoring and detecting environmental toxicants. In Escherichia coli, the multiple antibiotic resistance regulator (MarR) induces transcription upon binding to salicylic acid (SA). We generated SA-specific E. coli cell-based bioreporters utilizing the operator region of the mar operon and MarR as components of the reporter and sensing domains, respectively. Although bioreporters based on endogenous MarR and wild-type E. coli cells responded to SA, their sensitivity and selectivity were insufficient for practical sample monitoring. To improve these parameters, we genetically engineered host strains for optimal MarR expression, which enhanced the sensitivity of the biosensor to micromolar quantities of SA with increased selectivity. Under the optimized experimental conditions, the biosensor could quantify SA in environmental samples. For validation, the SA concentration in artificially contaminated SA-containing cosmetic samples was determined using the developed biosensor. Reliability assessment by comparing the concentrations determined using LC-MS/MS revealed > 90% accuracy of the bioreporters. Although bioreporters are not considered standard tools for environmental monitoring, bacterial cell-based bioreporters may serve as alternative tools owing to their affordability and simplicity. The SA biosensor developed in this study can potentially be a valuable tool for monitoring SA in environmental systems. KEY POINTS: • SA-responsive bioreporter is generated by employing mar operon system in E. coli • SA specificity and selectivity were enhanced by genetic/biochemical engineering • The novel bioreporter would be valuable for SA monitoring in environmental systems.

PEP-ASSOCIATED PROTEIN 3 regulates rice tiller formation and grain yield by controlling chloroplast biogenesis.

Plastid-encoded RNA polymerase (PEP) plays a pivotal role in chloroplast development by governing the transcription of chloroplast genes, and PEP-associated proteins (PAPs) modulate PEP transcriptional activity. Therefore, PAPs provide an intriguing target for those efforts to improve yield, by enhancing chloroplast development. In this study, we identified the rice (Oryza sativa) OsPAP3 gene and characterized its function in chloroplast development. OsPAP3 expression was light-dependent and leaf-specific, similar to the PEP-dependent chloroplast gene RUBISCO LARGE SUBUNIT (OsRbcL), and OsPAP3 protein localized to chloroplast nucleoids where PEP functions. Analysis of loss-of-function and gain-of-function mutants showed that the expression of OsPAP3 is tightly linked to chloroplast gene expression and chloroplast biogenesis in rice. Homozygous knockout mutants of OsPAP3 had fewer chloroplasts than wild type, whereas plants overexpressing OsPAP3 had more chloroplasts. Also, OsPAP3 knockout suppressed the PEP-dependent expression of chloroplast genes, but OsPAP3 overexpression increased their expression. These findings indicate that OsPAP3 regulates chloroplast biogenesis in rice by controlling the PEP-dependent expression of chloroplast genes. More importantly, data from 3 seasons of field cultivation revealed that the overexpression of OsPAP3 improves rice grain yield by approximately 25%, largely due to increased tiller formation. Collectively, these observations suggest that OsPAP3 regulates rice growth and productivity by promoting chloroplast development.

BML-281 promotes neuronal differentiation by modulating Wnt/Ca2+ and Wnt/PCP signaling pathway.

Histone deacetylase (HDAC) inhibitors promote differentiation through post-translational modifications of histones. BML-281, an HDAC6 inhibitor, has been known to prevent tumors, acute dextran sodium sulfate-associated colitis, and lung injury. However, the neurogenic differentiation effect of BML-281 is poorly understood. In this study, we investigated the effect of BML-281 on neuroblastoma SH-SY5Y cell differentiation into mature neurons by immunocytochemistry (ICC), reverse transcriptase PCR (RT-PCR), quantitative PCR (qPCR), and western blotting analysis. We found that the cells treated with BML-281 showed neurite outgrowth and morphological changes into mature neurons under a microscope. It was confirmed that the gene expression of neuronal markers (NEFL, MAP2, Tuj1, NEFH, and NEFM) was increased with certain concentrations of BML-281. Similarly, the protein expression of neuronal markers (NeuN, Synaptophysin, Tuj1, and NFH) was upregulated with BML-281 compared to untreated cells. Following treatment with BML-281, the expression of Wnt5α increased, and downstream pathways were activated. Interestingly, both Wnt/Ca2+ and Wnt/PCP pathways activated and regulated PKC, Cdc42, RhoA, Rac1/2/3, and p-JNK. Therefore, BML-281 induces the differentiation of SH-SY5Y cells into mature neurons by activating the non-canonical Wnt signaling pathway. From these results, we concluded that BML-281 might be a novel drug to differentiation into neuronal cells through the regulation of Wnt signaling pathway to reduce the neuronal cell death.

High-throughput discovery of plastid genes causing albino phenotypes in ornamental chimeric plants.

Chimeric plants composed of green and albino tissues have great ornamental value. To unveil the functional genes responsible for albino phenotypes in chimeric plants, we inspected the complete plastid genomes (plastomes) in green and albino leaf tissues from 23 ornamental chimeric plants belonging to 20 species, including monocots, dicots, and gymnosperms. In nine chimeric plants, plastomes were identical between green and albino tissues. Meanwhile, another 14 chimeric plants were heteroplasmic, showing a mutation between green and albino tissues. We identified 14 different point mutations in eight functional plastid genes related to plastid-encoded RNA polymerase (rpo) or photosystems which caused albinism in the chimeric plants. Among them, 12 were deleterious mutations in the target genes, in which early termination appeared due to small deletion-mediated frameshift or single nucleotide substitution. Another was single nucleotide substitution in an intron of the ycf3 and the other was a missense mutation in coding region of the rpoC2 gene. We inspected chlorophyll structure, protein functional model of the rpoC2, and expression levels of the related genes in green and albino tissues of Reynoutria japonica. A single amino acid change, histidine-to-proline substitution, in the rpoC2 protein may destabilize the peripheral helix of plastid-encoded RNA polymerase, impairing the biosynthesis of the photosynthesis system in the albino tissue of R. japonica chimera plant.

Division of cortical cells is regulated by auxin in Arabidopsis roots.

The root cortex transports water and nutrients absorbed by the root epidermis into the vasculature and stores substances such as starch, resins, and essential oils. The cortical cells are also deeply involved in determining epidermal cell fate. In Arabidopsis thaliana roots, the cortex is composed of a single cell layer generated by a single round of periclinal division of the cortex/endodermis initials. To further explore cortex development, we traced the development of the cortex by counting cortical cells. Unlike vascular cells, whose number increased during the development of root apical meristem (RAM), the number of cortical cells did not change, indicating that cortical cells do not divide during RAM development. However, auxin-induced cortical cell division, and this finding was confirmed by treatment with the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) and examining transgenic plants harboring CO2::ΔARF5, in which cortical expression of truncated AUXIN RESPONSE FACTOR5 (ΔARF5) induces auxin responses. NPA-induced cortical auxin accumulation and CO2::ΔARF5-mediated cortical auxin response induced anticlinal and periclinal cell divisions, thus increasing the number of cortical cells. These findings reveal a tight link between auxin and cortical cell division, suggesting that auxin is a key player in determining root cortical cell division.

Transcription Factor-Based Biosensors for Detecting Pathogens.

Microorganisms are omnipresent and inseparable from our life. Many of them are beneficial to humans, while some are not. Importantly, foods and beverages are susceptible to microbial contamination, with their toxins causing illnesses and even death in some cases. Therefore, monitoring and detecting harmful microorganisms are critical to ensuring human health and safety. For several decades, many methods have been developed to detect and monitor microorganisms and their toxicants. Conventionally, nucleic acid analysis and antibody-based analysis were used to detect pathogens. Additionally, diverse chromatographic methods were employed to detect toxins based on their chemical and structural properties. However, conventional techniques have several disadvantages concerning analysis time, sensitivity, and expense. With the advances in biotechnology, new approaches to detect pathogens and toxins have been reported to compensate for the disadvantages of conventional analysis from different research fields, including electrochemistry, nanotechnology, and molecular biology. Among them, we focused on the recent studies of transcription factor (TF)-based biosensors to detect microorganisms and discuss their perspectives and applications. Additionally, the other biosensors for detecting microorganisms reported in recent studies were also introduced in this review.

Design of Pb(II)-Specific E. coli-Based Biosensors by Engineering Regulatory Proteins and Host Cells.

Bacterial cell-based biosensors have been widely developed for detecting environmental toxic materials. The znt-operon in Escherichia coli is a Zn(II)-responsive genetic system and is employed in Zn(II), Cd(II), and Hg(II)-sensing biosensors. In this study, point mutations were introduced in the regulatory protein ZntR to modulate its target selectivity, and metal ion-exporting genes, such as copA and zntA, in host cells were deleted to increase cellular metal ion levels and enhance specificity. Thus, the overall responses of the E. coli cell-based biosensors toward metal(loid) ions were increased, and their selectivity, which was originally for Cd(II) and Hg(II), was shifted to Pb(II). The gene encoding ZntA, known as the Zn(II)-translocating P-type ATPase, showed an impact on the ability of E. coli to export Pb(II), whereas copA deletion showed no significant impact. Noteworthily, the newly generated biosensors employing ZntR Cys115Ile showed the capacity to detect under 5 nM Pb(II) in solution, without response to other tested metal ions within 0-100 nM. To understand the marked effect of single point mutations on ZntR, computational modeling was employed. Although it did not provide clear answers, changes in the sequences of the metal-binding loops of ZntR modulated its transcriptional strength and target selectivity. In summary, the approaches proposed in this study can be valuable to generate new target-sensing biosensors with superior selectivity and specificity, which can in turn broaden the applicability of cell-based biosensors to monitor Pb(II) in environmental systems.

Development of novel Escherichia coli cell-based biosensors to monitor Mn(II) in environmental systems.

Escherichia coli uses manganese [Mn(II)] as an essential trace element; thus, it has a genetic system that regulates cellular Mn(II) levels. Several genes in the mnt-operon of E. coli respond to intercellular Mn(II) levels, and transcription is regulated by a transcription factor (MntR) that interacts with Mn(II). This study aimed to develop Mn(II)-sensing biosensors based on mnt-operon genetic systems. Additionally, the properties of biosensors developed based on the promoter regions of mntS, mntH, and mntP were investigated. MntR represses the transcription of MntS and MntH after binding with Mn(II), while it induces MntP transcription. Thus, Mn(II) biosensors that decrease and increase signals could be obtained by fusing the promoter regions of mntS/mntH and mntP, with egfp encoding an enhanced green fluorescent protein. However, only the biosensor-based mntS:egfp responded to Mn(II) exposure. Further, E. coli harboring P mntS :egfp showed a concentration-dependent decrease in fluorescence signals. To enhance the sensitivity of the biosensor toward Mn(II), E. coli containing a deleted MntP gene that encodes Mn(II) exporter, was used as a host cell for biosensor development. The sensitivity toward Mn(II) increased by two times on using E. coli-mntP, and the biosensor could quantify 0.01-10 µM of Mn(II). Further, the applicability of Mn(II) in artificially contaminated water samples was quantified and showed >95% accuracy. The newly developed Mn(II) biosensors could detect and quantify the residual Mn(II) from mancozeb in soil samples, with the quantification accuracy being approximately 90%. To the best of our knowledge, this is the first Mn (II)-specific bacterial cell-based biosensor that serves as a valuable tool for monitoring and assessing the risks of Mn(II) in environmental systems.

Isolation of high-purity and high-stability exosomes from ginseng.

Exosomes are nano-sized extracellular vesicles that regulate cell growth and defense by delivering bioactive cellular constituents. They are a promising material for biomedical and cosmetic utilization, especially in medicinal crops such as ginseng. One main hurdle to their usage is the need for a method to isolate stable exosomes with high purity. In this study, we first tested two methods to isolate exosomes from ginseng: ultracentrifugation, the most widely used method; and the ExoQuick system, a polymer-based exosome precipitation approach. We also designed and tested a third method in which we combined ultracentrifugation and ExoQuick methods. Size distribution analysis revealed that the exosome isolation purity by the ultracentrifugation and ExoQuick methods alone were 34.1% and 59.7%, respectively, while the combination method greatly improved exosome isolation purity (83.3%). Furthermore, we found that the combination method also increases the colloidal stability of isolated ginseng exosomes, and the increase was almost double that of the ultracentrifugation method. Lastly, we showed that the combination method can also be used to isolate high-purity and high-stability exosomes from the model plant Arabidopsis. Overall, our findings indicate that the combination method is suitable to isolate high-purity and high-stability exosomes from plants including ginseng.

Auxin controls the division of root endodermal cells.

The root endodermis forms a selective barrier that prevents the free diffusion of solutes into the vasculature; to make this barrier, endodermal cells deposit hydrophobic compounds in their cell walls, forming the Casparian strip. Here, we showed that, in contrast to vascular and epidermal root cells, endodermal root cells do not divide alongside the root apical meristem in Arabidopsis thaliana. Auxin treatment induced division of endodermal cells in wild-type plants, but not in the auxin signaling mutant auxin resistant3-1. Endodermis-specific activation of auxin responses by expression of truncated AUXIN-RESPONSIVE FACTOR5 (ΔARF5) in root endodermal cells under the control of the ENDODERMIS7 promoter (EN7::ΔARF5) also induced endodermal cell division. We used an auxin transport inhibitor to cause accumulation of auxin in endodermal cells, which induced endodermal cell division. In addition, knockout of P-GLYCOPROTEIN1 (PGP1) and PGP19, which mediate centripetal auxin flow, promoted the division of endodermal cells. Together, these findings reveal a tight link between the endodermal auxin response and endodermal cell division, suggesting that auxin is a key regulator controlling the division of root endodermal cells, and that PGP1 and PGP19 are involved in regulating endodermal cell division.

Promoted ABA Hydroxylation by Capsicum annuum CYP707As Overexpression Suppresses Pollen Maturation in Nicotiana tabacum.

Abscisic acid (ABA) is a key signaling molecule that mediates plant response to stress. Increasing evidence indicates that ABA also regulates many aspects of plant development, such as seed germination, leaf development, and ripening. ABA metabolism, including ABA biosynthesis and degradation, is an essential aspect of ABA response in plants. In this study, we identified four cytochrome P450 genes (CaCYP707A1, 2, 3, and 4) that mediate ABA hydroxylation, which is required for ABA degradation in Capsicum annuum. We observed that CaCYP707A-mediated ABA hydroxylation promotes ABA degradation, leading to low levels of ABA and a dehydration phenotype in 35S:CaCYP707A plants. Importantly, seed formation was strongly inhibited in 35S:CaCYP707A plants, and a cross-pollination test suggested that the defect in seed formation is caused by improper pollen development. Phenotypic analysis showed that pollen maturation is suppressed in 35S:CaCYP707A1 plants. Consequently, most 35S:CaCYP707A1 pollen grains degenerated, unlike non-transgenic (NT) pollen, which developed into mature pollen grains. Together our results indicate that CaCYP707A mediates ABA hydroxylation and thereby influences pollen development, helping to elucidate the mechanism underlying ABA-regulated pollen development.

Environmental Signal-Dependent Regulation of Flowering Time in Rice.

The transition from the vegetative to the reproductive stage of growth is a critical event in the lifecycle of a plant and is required for the plant's reproductive success. Flowering time is tightly regulated by an internal time-keeping system and external light conditions, including photoperiod, light quality, and light quantity. Other environmental factors, such as drought and temperature, also participate in the regulation of flowering time. Thus, flexibility in flowering time in response to environmental factors is required for the successful adaptation of plants to the environment. In this review, we summarize our current understanding of the molecular mechanisms by which internal and environmental signals are integrated to regulate flowering time in Arabidopsis thaliana and rice (Oryza sativa).

Shifting the Specificity of E. coli Biosensor from Inorganic Arsenic to Phenylarsine Oxide through Genetic Engineering.

It has recently been discovered that organic and inorganic arsenics could be detrimental to human health. Although organic arsenic is less toxic than inorganic arsenic, it could form inorganic arsenic through chemical and biological processes in environmental systems. In this regard, the availability of tools for detecting organic arsenic species would be beneficial. Because As-sensing biosensors employing arsenic responsive genetic systems are regulated by ArsR which detects arsenics, the target selectivity of biosensors could be obtained by modulating the selectivity of ArsR. In this study, we demonstrated a shift in the specificity of E. coli cell-based biosensors from the detection of inorganic arsenic to that of organic arsenic, specifically phenylarsine oxide (PAO), through the genetic engineering of ArsR. By modulating the number and location of cysteines forming coordinate covalent bonds with arsenic species, an E. coli cell-based biosensor that was specific to PAO was obtained. Despite its restriction to PAO at the moment, it offers invaluable evidence of the potential to generate new biosensors for sensing organic arsenic species through the genetic engineering of ArsR.

Modulation of the Metal(loid) Specificity of Whole-Cell Bioreporters by Genetic Engineering of ZntR Metal-Binding Loops.

Bacterial cell-based biosensors, or whole-cell bioreporters (WCBs), are an alternative tool for the quantification of hazardous materials. Most WCBs share similar working mechanisms. In brief, the recognition of a target by sensing domains induces a biological event, such as changes in protein conformation or gene expression, providing a basis for quantification. WCBs targeting heavy metal(loid)s employ metalloregulators as sensing domains and control the expression of genes in the presence of target metal(loid) ions, but the diversity of targets, specificity, and sensitivity of these WCBs are limited. In this study, we genetically engineered the metal-binding loop (MBL) of ZntR, which controls the znt-operon in Escherichia coli. In the MBL of ZntR, three Cys sites interact with metal ions. Based on the crystal structure of ZntR, MBL sequences were modified by sitedirected mutagenesis. As a result, the metal-sensing properties of WCBs differed depending on amino acid sequences and the new selectivity to Cr or Pb was observed. Although there is room for improvement, our results support the use of currently available WCBs as a platform to generate new WCBs to target other environmental pollutants including metal(loid)s.

Root Development and Stress Tolerance in rice: The Key to Improving Stress Tolerance without Yield Penalties.

Roots anchor plants and take up water and nutrients from the soil; therefore, root development strongly affects plant growth and productivity. Moreover, increasing evidence indicates that root development is deeply involved in plant tolerance to abiotic stresses such as drought and salinity. These findings suggest that modulating root growth and development provides a potentially useful approach to improve plant abiotic stress tolerance. Such targeted approaches may avoid the yield penalties that result from growth-defense trade-offs produced by global induction of defenses against abiotic stresses. This review summarizes the developmental mechanisms underlying root development and discusses recent studies about modulation of root growth and stress tolerance in rice.

SHORTROOT-Mediated Intercellular Signals Coordinate Phloem Development in Arabidopsis Roots.

Asymmetric cell division (ACD) and positional signals play critical roles in the tissue patterning process. In the Arabidopsis (Arabidopsis thaliana) root meristem, two major phloem cell types arise via ACDs of distinct origins: one for companion cells (CCs) and the other for proto- and metaphloem sieve elements (SEs). The molecular mechanisms underlying each of these processes have been reported; however, how these are coordinated has remained elusive. Here, we report a new phloem development process coordinated via the SHORTROOT (SHR) transcription factor in Arabidopsis. The movement of SHR into the endodermis regulates the ACD for CC formation by activating microRNA165/6, while SHR moving into the phloem regulates the ACD generating the two phloem SEs. In the phloem, SHR sequentially activates NAC-REGULATED SEED MORPHOLOGY 1 (NARS1) and SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN 2 (SND2), and these three together form a positive feedforward loop. Under this regulatory scheme, NARS1, generated in the CCs of the root differentiation zone, establishes a top-down signal that drives the ACD for phloem SEs in the meristem. SND2 appears to function downstream to amplify NARS1 via positive feedback. This new regulatory mechanism expands our understanding of the sophisticated vascular tissue patterning processes occurring during postembryonic root development.plantcell;32/5/1519/FX1F1fx1.

Antimony sensing whole-cell bioreporters derived from ArsR genetic engineering.

Despite the known hazardous effects of antimony (Sb) on human health, Sb monitoring biosensors have not been as actively investigated as arsenic (As) biosensors. Whole-cell bioreporters (WCBs) employing an arsenic-responsive operon and a regulatory protein (ArsR) are reportedly capable of monitoring arsenite, arsenate, and antimonite. However, the potential of WCBs as Sb biosensors has been largely ignored. Here, the metal-binding site of ArsR (sequenced as ELCVCDLCTA from amino acid number 30 to 39) was modified via genetic engineering to enhance Sb specificity. By relocating cysteine residues and introducing point mutations, nine ArsR mutants were generated and tested for metal(loid) ion specificity. The Sb specificity of WCBs was enhanced by the C37S/A39C and L36C/C37S mutations on the As binding site of ArsR. Additionally, WCBs with other ArsR mutants exhibited new target sensing capabilities toward Cd and Pb. Although further research is required to enhance the specificity and sensitivity of WCBs and to broaden their practical applications, our proposed strategy based on genetic engineering of regulatory proteins provides a valuable basis to generate WCBs to monitor novel targets.

Crosstalk with Jasmonic Acid Integrates Multiple Responses in Plant Development.

To date, extensive studies have identified many classes of hormones in plants and revealed the specific, nonredundant signaling pathways for each hormone. However, plant hormone functions largely overlap in many aspects of plant development and environmental responses, suggesting that studying the crosstalk among plant hormones is key to understanding hormonal responses in plants. The phytohormone jasmonic acid (JA) is deeply involved in the regulation of plant responses to biotic and abiotic stresses. In addition, a growing number of studies suggest that JA plays an essential role in the modulation of plant growth and development under stress conditions, and crosstalk between JA and other phytohormones involved in growth and development, such as gibberellic acid (GA), cytokinin, and auxin modulate various developmental processes. This review summarizes recent findings of JA crosstalk in the modulation of plant growth and development, focusing on JA-GA, JA-cytokinin, and JA-auxin crosstalk. The molecular mechanisms underlying this crosstalk are also discussed.

Specific heavy metal/metalloid sensors: current state and perspectives.

Heavy metal(loid)s play pivotal roles in regulating physiological and developmental aspects in living organisms depending on their concentration. For example, a trace amount of heavy metal(loid)s is essential for living organisms, but heavy metal(loid)s in high concentrations negatively affect their physiology and development. Because of rapid industrial developments, heavy metal(loid)s have been accumulating in environmental systems, thereby becoming a threat to human health and the earth's ecosystem. Thus, the development of tools to quantify and monitor heavy metal(loid)s in environmental systems has become essential. Typically, risk has been determined through instrument-based analysis, regardless of the shortcomings regarding expense and duration. Nowadays, the need for alternative tools, besides instrumental analysis, to detect heavy metals has prompted the development of new techniques, and many different methods have been reported from various research areas, including new techniques based on electrochemistry and biological systems. Nonetheless, it seems that the gap between laboratory and fieldwork is still greater than it should be when it comes to applying these systems. In this mini-review, we discuss the current status of heavy metals/metalloid detection techniques, with an emphasis on biosensors. Moreover, we discuss the advantages and disadvantages as well as the mechanisms behind newly developed sensors and make suggestions to improve applicability and to develop new objective targeting sensors. Although many different types of metal(loid) sensors are available, we focused on metal sensors based on biological systems. Additionally, we suggest potent approaches to developing new biosensor systems based on current metal sensor mechanisms.

An isoform of the plastid RNA polymerase-associated protein FSD3 negatively regulates chloroplast development.

BACKGROUND: Plastid-encoded RNA polymerase (PEP) plays an essential role in chloroplast development by governing the expression of genes involved in photosynthesis. At least 12 PEP-associated proteins (PAPs), including FSD3/PAP4, regulate PEP activity and chloroplast development by modulating formation of the PEP complex. RESULTS: In this study, we identified FSD3S, a splicing variant of FSD3; the FSD3 and FSD3S transcripts encode proteins with identical N-termini, but different C-termini. Characterization of FSD3 and FSD3S proteins showed that the C-terminal region of FSD3S contains a transmembrane domain, which promotes FSD3S localization to the chloroplast membrane but not to nucleoids, in contrast to FSD3, which localizes to the chloroplast nucleoid. We also found that overexpression of FSD3S negatively affects photosynthetic activity and chloroplast development by reducing expression of genes involved in photosynthesis. In addition, FSD3S failed to complement the chloroplast developmental defects in the fsd3 mutant. CONCLUSION: These results suggest FSD3 and FSD3S, with their distinct localization patterns, have different functions in chloroplast development, and FSD3S negatively regulates expression of PEP-dependent chloroplast genes, and development of chloroplasts.