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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

pTAC10, a Key Subunit of Plastid-Encoded RNA Polymerase, Promotes Chloroplast Development.

Regulation of photosynthetic gene expression by plastid-encoded RNA polymerase (PEP) is essential for chloroplast development. The activity of PEP largely relies on at least 12 PEP-associated proteins (PAPs) encoded in the nuclear genome of plant cells. A recent model proposed that these PAPs regulate the establishment of the PEP complex through broad PAP-PEP or PAP-PAP interactions. In this study, we identified the Arabidopsis (Arabidopsis thaliana) seedling-lethal mutant ptac10-1, which has defects in chloroplast development, and found that the mutant phenotype is caused by the suppression of PLASTID S1 RNA-BINDING DOMAIN PROTEIN (pTAC10/PAP3). Analysis of the heterozygous mutant and pTAC10-overexpressing transgenic plants indicated that the expression level of pTAC10 is tightly linked to chloroplast development. Characterization of the interaction of pTAC10 with PAPs revealed that pTAC10 interacts with other PAPs, such as FSD2, FSD3, TrxZ, pTAC7, and pTAC14, but it does not interact with PEP core enzymes, such as rpoA and rpoB. Analysis of pTAC10 interactions using truncated pTAC10 proteins showed that the pTAC10 carboxyl-terminal region downstream of the S1 domain is involved in the pTAC10-PAP interaction. Furthermore, overexpression of truncated pTAC10s lacking the C-terminal regions downstream of the S1 domain could not rescue the ptac10-1 mutant phenotype and induced an abnormal whitening phenotype in Columbia-0 plants. Our observations suggested that these pTAC10-PAP interactions are essential for the formation of the PEP complex and chloroplast development.

Modulating the sensing properties of Escherichia coli-based bioreporters for cadmium and mercury.

Despite the large number of bioreporters developed to date, the ability to detect heavy metal(loid)s with bioreporters has thus far been limited owing to the lack of appropriate genetic systems. We here present a novel approach to modulate the selectivity and sensitivity of microbial whole-cell bioreporters (WCBs) for sensing metal(loid)s via the znt-operon from Escherichia coli, which were applied to quantify the bioavailability of these contaminants in environmental samples. The WCB harboring the fusion gene zntAp::egfp was used as a microbial metal(loid) sensor, which was turned on by the interaction between ZntR and metal(loid) ions. This design makes it possible to modulate the selectivity and sensitivity to metal(loid)s simply by changing the metal-binding property of ZntR and by disrupting the metal efflux system of E. coli, respectively. In fact, the E. coli cell-based bioreporter harboring zntAp::egfp showed multi-target responses to Cd(II), Hg(II), and Zn(II). However, the WCBs showed responses toward only Cd(II) and Hg(II) when the amino acid sequence of the metal-binding loop of ZntR was changed to CNHEPGTVCPIC and CPGDDSADC, respectively. Moreover, the sensitivity toward both Cd(II) and Hg(II) was enhanced when copA, which is known to export copper and silver, was deleted. Thus, our findings provide a strong foundation for expanding the target of WCBs from the currently limited number of genetic systems available.

Enhancing the copper-sensing capability of Escherichia coli-based whole-cell bioreporters by genetic engineering.

Metals are essential to all organisms; accordingly, cells employ numerous genes to maintain metal homeostasis as high levels can be toxic. In the present study, the gene operons responsive to metal(loid)s were employed to generate bacterial cell-based biosensors to detect target metal(loid)s. The cluster of genes related to copper transport known as the cop-operon is regulated by the interaction between the copA promoter region (copAp) and CueR, turning on and off gene expression upon copper ion binding. Therefore, the detection of copper ions could be achieved by inserting a plasmid harboring the fusion of copAp and reporter genes, such as enzymes and fluorescent genes. However, copAp is not as strong a promoter as other metal-inducible promoters, such as znt-, mer-, and ars-operons; thereby, its sensitivity toward copper ions was not sufficient for quantification. To overcome this problem, we engineered Escherichia coli with a deletion of copA to interfere with copper export from cells. The engineered E. coli whole-cell bioreporter was able to detect copper ions at 0 to 10 µM in an aqueous solution. Most importantly, it was specific to copper among several tested heavy metal(loid)s. Therefore, it will likely be useful to detect copper in diverse environmental systems. Although additional improvements are still required to optimize the E. coli-based copper-sensing whole-cell bioreporters presented in this study, our results suggest that there is huge potential to generate whole-cell bioreporters for additional targets by molecular engineering.

The rice OsNAC6 transcription factor orchestrates multiple molecular mechanisms involving root structural adaptions and nicotianamine biosynthesis for drought tolerance.

Drought has a serious impact on agriculture worldwide. A plant's ability to adapt to rhizosphere drought stress requires reprogramming of root growth and development. Although physiological studies have documented the root adaption for tolerance to the drought stress, underlying molecular mechanisms is still incomplete, which is essential for crop engineering. Here, we identified OsNAC6-mediated root structural adaptations, including increased root number and root diameter, which enhanced drought tolerance. Multiyear drought field tests demonstrated that the grain yield of OsNAC6 root-specific overexpressing transgenic rice lines was less affected by drought stress than were nontransgenic controls. Genome-wide analyses of loss- and gain-of-function mutants revealed that OsNAC6 up-regulates the expression of direct target genes involved in membrane modification, nicotianamine (NA) biosynthesis, glutathione relocation, 3'-phophoadenosine 5'-phosphosulphate accumulation and glycosylation, which represent multiple drought tolerance pathways. Moreover, overexpression of NICOTIANAMINE SYNTHASE genes, direct targets of OsNAC6, promoted the accumulation of the metal chelator NA and, consequently, drought tolerance. Collectively, OsNAC6 orchestrates novel molecular drought tolerance mechanisms and has potential for the biotechnological development of high-yielding crops under water-limiting conditions.

Overexpression of the OsERF71 Transcription Factor Alters Rice Root Structure and Drought Resistance.

Plant responses to drought stress require the regulation of transcriptional networks via drought-responsive transcription factors, which mediate a range of morphological and physiological changes. AP2/ERF transcription factors are known to act as key regulators of drought resistance transcriptional networks; however, little is known about the associated molecular mechanisms that give rise to specific morphological and physiological adaptations. In this study, we functionally characterized the rice (Oryza sativa) drought-responsive AP2/ERF transcription factor OsERF71, which is expressed predominantly in the root meristem, pericycle, and endodermis. Overexpression of OsERF71, either throughout the entire plant or specifically in roots, resulted in a drought resistance phenotype at the vegetative growth stage, indicating that overexpression in roots was sufficient to confer drought resistance. The root-specific overexpression was more effective in conferring drought resistance at the reproductive stage, such that grain yield was increased by 23% to 42% over wild-type plants or whole-body overexpressing transgenic lines under drought conditions. OsERF71 overexpression in roots elevated the expression levels of genes related to cell wall loosening and lignin biosynthetic genes, which correlated with changes in root structure, the formation of enlarged aerenchyma, and high lignification levels. Furthermore, OsERF71 was found to directly bind to the promoter of OsCINNAMOYL-COENZYME A REDUCTASE1, a key gene in lignin biosynthesis. These results indicate that the OsERF71-mediated drought resistance pathway recruits factors involved in cell wall modification to enable root morphological adaptations, thereby providing a mechanism for enhancing drought resistance.

Cytokinin-dependent secondary growth determines root biomass in radish (Raphanus sativus L.).

The root serves as an essential organ in plant growth by taking up nutrients and water from the soil and supporting the rest of the plant body. Some plant species utilize roots as storage organs. Sweet potatoes (Ipomoea batatas), cassava (Manihot esculenta), and radish (Raphanus sativus), for example, are important root crops. However, how their root growth is regulated remains unknown. In this study, we characterized the relationship between cambium and radial root growth in radish. Through a comparative analysis with Arabidopsis root expression data, we identified putative cambium-enriched transcription factors in radish and analysed their expression in representative inbred lines featuring distinctive radial growth. We found that cell proliferation activities in the cambium positively correlated with radial growth and final yields of radish roots. Expression analysis of candidate transcription factor genes revealed that some genes are differentially expressed between inbred lines and that the difference is due to the distinct cytokinin response. Taken together, we have demonstrated for the first time, to the best of our knowledge, that cytokinin-dependent radial growth plays a key role in the yields of root crops.

RSL genes are sufficient for rhizoid system development in early diverging land plants.

Land plants are anchored to their substratum from which essential inorganic nutrients are taken up. These functions are carried out by a system of rhizoids in early diverging groups of land plants, such as mosses, liverworts and hornworts. Physcomitrella patens RHD SIX-LIKE1 (PpRSL1) and PpRSL2 transcription factors are necessary for rhizoid development in mosses. Similar proteins, AtRHD6 and AtRSL1, control the development of root hairs in Arabidopsis thaliana. Auxin positively regulates root hair development independently of AtRHD6 and AtRSL1 in A. thaliana but the regulatory interactions between auxin and PpRSL1 and PpRSL2 are unknown. We show here that co-expression of PpRSL1 and PpRSL2 is sufficient for the development of the rhizoid system in the moss P. patens; constitutive expression of PpRSL1 and PpRSL2 converts developing leafy shoot axes (gametophores) into rhizoids. During wild-type development, PpRSL1 and PpRSL2 are expressed in the specialized cells that develop rhizoids, indicating that cell-specific expression of PpRSL1 and PpRSL2 is sufficient to promote rhizoid differentiation during wild-type P. patens development. In contrast to A. thaliana, auxin promotes rhizoid development by positively regulating PpRSL1 and PpRSL2 activity in P. patens. This indicates that even though the same genes control the development of root hairs and rhizoids, the regulation of this transcriptional network by auxin is different in these two species. This suggests that auxin might have controlled the development of the first land plant soil anchoring systems that evolved 465 million years ago by regulating the expression of RSL genes and that this regulatory network has changed since mosses and angiosperms last shared a common ancestor.

Intercellular trafficking of transcription factors in the vascular tissue patterning.

Throughout life cycles, plants grow in an indeterminate manner by adding new cells and organs with specialized functions. Newly emerging cells acquire their identities depending on their positions relative to the neighboring cells. Exchanging positional signals between cells is critical in this process. Recent studies showed that many transcription factors move between cells or between organs in forms of proteins and messenger RNA (mRNA). Some of these were found to be important positional signals for cell type patterning. Cell type patterning in the vascular system is no exception from this. In this review, we describe recent discoveries of mobile transcription factors that function as positional signals for vascular tissue patterning and propose how these transcription factors integrate with other forms of signals.