scholarly journals Fluorescent reporters for functional analysis in rice leaves

2019 ◽  
Author(s):  
Leonie H. Luginbuehl ◽  
Sherif El-Sharnouby ◽  
Na Wang ◽  
Julian M. Hibberd
Keyword(s):  
Gene Expression ◽  
Functional Analysis ◽  
Light Scattering ◽  
Leaf Blade ◽  
Fluorescent Proteins ◽  
Protein Localization ◽  
Fluorescent Reporters ◽  
Microparticle Bombardment ◽  
Rice Leaf ◽  
Rice Leaves

AbstractFluorescent reporters have facilitated non-invasive imaging in multiple plant species and thus allowed analysis of processes ranging from gene expression and protein localization through to cellular patterning. However, in rice, a globally important crop and model species, there are relatively few reports of fluorescent proteins being used in leaves. Fluorescence imaging is particularly difficult in the rice leaf blade, likely due to a high degree of light scattering in this tissue. To address this, we investigated approaches to improve deep imaging in mature rice leaf blades. We found that ClearSee treatment, which has previously been used to visualise fluorescent reporters in whole tissues of plants, led to improved imaging in rice. Removing epidermal and subtending mesophyll cell layers was faster than ClearSee, and also reduced light scattering such that imaging of fluorescent proteins in deeper leaf layers was possible. To expand the range of fluorescent proteins suitable for imaging in rice, we screened twelve whose spectral profiles spanned most of the visible spectrum. This identified five proteins, mTurquoise2, mClover3, mNeonGreen, mKOκ and tdTomato that are robustly expressed and visible in mesophyll cells of stably transformed plants. Using microparticle bombardment, we show that mTurquoise2 and mNeonGreen can be used for simultaneous multicolour imaging of different sub-cellular compartments. Overall, we conclude that mTurquoise2, mClover3, mNeonGreen, mKOκ and tdTomato are suitable for high resolution live imaging of rice leaves, both after transient and stable transformation. Along with the rapid microparticle bombardment method, which allows transient transformation of major cell types in the leaf blade, these fluorescent reporters should greatly facilitate the analysis of gene expression and cell biology in rice.One sentence summaryWe report five fluorescent reporters suitable for functional analysis in rice leaves.

scholarly journals Genome-Wide Analysis of Spatiotemporal Expression Patterns During Rice Leaf Development

2020 ◽  
Author(s):  
Masayuki Miya ◽  
Takanori Yoshikawa ◽  
Yutaka Sato ◽  
Jun-Ichi Itoh
Keyword(s):  
Leaf Development ◽  
Leaf Blade ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Gene Clusters ◽  
Mature Stage ◽  
Future Research ◽  
Rice Leaf ◽  
Genome Wide ◽  
Rice Leaves

Abstract Background: Rice leaves consist of three distinct regions along a proximal-distal axis, namely the leaf blade, sheath, and blade-sheath boundary region. Each region has a unique morphology and function, but the genetic programs underlying the development of each region are poorly understood. To fully elucidate rice leaf development and discover genes with unique functions in rice and grasses, it is crucial to explore genome-wide transcriptional profiles during the development of the three regions.Results: In this study, we performed microarray analysis to profile the spatial and temporal patterns of gene expression in the rice leaf using dissected parts of leaves sampled in broad developmental stages. The dynamics in each region revealed that the transcriptomes changed dramatically throughout the progress of tissue differentiation, and those of the leaf blade and sheath differed greatly at the mature stage. Cluster analysis of expression patterns among leaf parts revealed groups of genes that may be involved in specific biological processes related to rice leaf development. Moreover, we found novel genes potentially involved in rice leaf development using a combination of transcriptome data and in situ hybridization, and analyzed their spatial expression patterns at high resolution. We successfully identified multiple genes that exhibit localized expression in tissues characteristic of rice or grass leaves.Conclusions: Although the genetic mechanisms of leaf development have been elucidated in several eudicots, direct application of that information to rice and grasses is not appropriate due to the morphological and developmental differences between them. Our analysis provides not only insights into the development of rice leaves but also expression profiles that serve as a valuable resource for gene discovery. The genes and gene clusters identified in this study may facilitate future research on the unique developmental mechanisms of rice leaves.

scholarly journals Genome-wide analysis of spatiotemporal expression patterns during rice leaf development

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Masayuki Miya ◽  
Takanori Yoshikawa ◽  
Yutaka Sato ◽  
Jun-Ichi Itoh
Keyword(s):  
Leaf Development ◽  
Leaf Blade ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Gene Clusters ◽  
Mature Stage ◽  
Future Research ◽  
Rice Leaf ◽  
Genome Wide ◽  
Rice Leaves

Abstract Background Rice leaves consist of three distinct regions along a proximal-distal axis, namely the leaf blade, sheath, and blade-sheath boundary region. Each region has a unique morphology and function, but the genetic programs underlying the development of each region are poorly understood. To fully elucidate rice leaf development and discover genes with unique functions in rice and grasses, it is crucial to explore genome-wide transcriptional profiles during the development of the three regions. Results In this study, we performed microarray analysis to profile the spatial and temporal patterns of gene expression in the rice leaf using dissected parts of leaves sampled in broad developmental stages. The dynamics in each region revealed that the transcriptomes changed dramatically throughout the progress of tissue differentiation, and those of the leaf blade and sheath differed greatly at the mature stage. Cluster analysis of expression patterns among leaf parts revealed groups of genes that may be involved in specific biological processes related to rice leaf development. Moreover, we found novel genes potentially involved in rice leaf development using a combination of transcriptome data and in situ hybridization, and analyzed their spatial expression patterns at high resolution. We successfully identified multiple genes that exhibit localized expression in tissues characteristic of rice or grass leaves. Conclusions Although the genetic mechanisms of leaf development have been elucidated in several eudicots, direct application of that information to rice and grasses is not appropriate due to the morphological and developmental differences between them. Our analysis provides not only insights into the development of rice leaves but also expression profiles that serve as a valuable resource for gene discovery. The genes and gene clusters identified in this study may facilitate future research on the unique developmental mechanisms of rice leaves.

scholarly journals Red Fluorescent Proteins for Gene Expression and Protein Localization Studies in Streptococcus pneumoniae and Efficient Transformation with DNA Assembled via the Gibson Assembly Method

2015 ◽  
Vol 81 (20) ◽  
pp. 7244-7252 ◽  
Author(s):  
Katrin Beilharz ◽  
Renske van Raaphorst ◽  
Morten Kjos ◽  
Jan-Willem Veening
Keyword(s):  
Gene Expression ◽  
Streptococcus Pneumoniae ◽  
Cell Biology ◽  
Fluorescent Proteins ◽  
Protein Localization ◽  
Gibson Assembly ◽  
Content Type ◽  
Assembly Method ◽  
Red Fluorescent Proteins ◽  
Wide Range

ABSTRACTDuring the last decades, a wide range of fluorescent proteins (FPs) have been developed and improved. This has had a great impact on the possibilities in biological imaging and the investigation of cellular processes at the single-cell level. Recently, we have benchmarked a set of green fluorescent proteins (GFPs) and generated a codon-optimized superfolder GFP for efficient use in the important human pathogenStreptococcus pneumoniaeand other low-GC Gram-positive bacteria. In the present work, we constructed and compared four red fluorescent proteins (RFPs) inS. pneumoniae. Two orange-red variants, mOrange2 and TagRFP, and two far-red FPs, mKate2 and mCherry, were codon optimized and examined by fluorescence microscopy and plate reader assays. Notably, protein fusions of the RFPs to FtsZ were constructed by direct transformation of linear Gibson assembly (isothermal assembly) products, a method that speeds up the strain construction process significantly. Our data show that mCherry is the fastest-maturing RFP inS. pneumoniaeand is best suited for studying gene expression, while mKate2 and TagRFP are more stable and are the preferred choices for protein localization studies. The RFPs described here will be useful for cell biology studies that require multicolor labeling inS. pneumoniaeand related organisms.

scholarly journals Use of mCherry Red Fluorescent Protein for Studies of Protein Localization and Gene Expression in Clostridium difficile

2014 ◽  
Vol 81 (5) ◽  
pp. 1652-1660 ◽  
Author(s):  
Eric M. Ransom ◽  
Craig D. Ellermeier ◽  
David S. Weiss
Keyword(s):  
Gene Expression ◽  
Clostridium Difficile ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Protein Localization ◽  
Anaerobic Bacteria ◽  
Gc Content ◽  
Inducible Promoter ◽  
Developed Countries ◽  
Content Type

ABSTRACTFluorescent proteins are powerful reporters in biology, but most require O2for chromophore maturation, making them inherently difficult to use in anaerobic bacteria.Clostridium difficile, a strict anaerobe with a genomic GC content of only 29%, is the leading cause of hospital-acquired diarrhea in developed countries, and new methods for studying this pathogen are sorely needed. We recently demonstrated that a cyan fluorescent protein called CFPoptthat has been codon optimized for production in low-GC bacteria can be used to study protein localization inC. difficileprovided the cells are fixed prior to exposure to air. We describe here a codon-optimized variant of mCherry (mCherryOpt) that exhibits faster acquisition of fluorescence and a better signal-to-noise ratio than CFPopt. We utilizedmCherryOptto construct plasmids for studying protein localization (pRAN473) and gene expression (pDSW1728) inC. difficile. Plasmid pRAN473 is anmCherryOptfusion vector with a tetracycline-inducible promoter. To document its biological utility, we demonstrated septal localization of two cell division proteins, MldA and ZapA. Plasmid pDSW1728 is designed for cloning a promoter of interest upstream ofmCherryOpt. As proof of principle, we studied the expression of thepdaVoperon, which is required for lysozyme resistance. In confirmation and extension of previous reports, we found that expression of thepdaVoperon requires the alternative sigma factor σvand that induction by lysozyme is dose dependent and uniform across the population of lysozyme-treated cells.

scholarly journals Fluorescent reporters for functional analysis in rice leaves

Plant Direct ◽  
2020 ◽  
Vol 4 (2) ◽  
Author(s):  
Leonie H. Luginbuehl ◽  
Sherif El‐Sharnouby ◽  
Na Wang ◽  
Julian M. Hibberd
Keyword(s):  
Functional Analysis ◽  
Fluorescent Reporters ◽  
Rice Leaves

scholarly journals Analysis of the transcriptome of bovine endometrial cells isolated by laser micro-dissection (1): specific signatures of stromal, glandular and luminal epithelial cells

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Wiruntita Chankeaw ◽  
Sandra Lignier ◽  
Christophe Richard ◽  
Theodoros Ntallaris ◽  
Mariam Raliou ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Localization ◽  
Expression Profiles ◽  
Cell Types ◽  
Molecular Signature ◽  
Receptor Protein ◽  
Specific Gene ◽  
Specific Cell ◽  
Biological Processes ◽  
Endometrial Cells

Abstract Background A number of studies have examined mRNA expression profiles of bovine endometrium at estrus and around the peri-implantation period of pregnancy. However, to date, these studies have been performed on the whole endometrium which is a complex tissue. Consequently, the knowledge of cell-specific gene expression, when analysis performed with whole endometrium, is still weak and obviously limits the relevance of the results of gene expression studies. Thus, the aim of this study was to characterize specific transcriptome of the three main cell-types of the bovine endometrium at day-15 of the estrus cycle. Results In the RNA-Seq analysis, the number of expressed genes detected over 10 transcripts per million was 6622, 7814 and 8242 for LE, GE and ST respectively. ST expressed exclusively 1236 genes while only 551 transcripts were specific to the GE and 330 specific to LE. For ST, over-represented biological processes included many regulation processes and response to stimulus, cell communication and cell adhesion, extracellular matrix organization as well as developmental process. For GE, cilium organization, cilium movement, protein localization to cilium and microtubule-based process were the only four main biological processes enriched. For LE, over-represented biological processes were enzyme linked receptor protein signaling pathway, cell-substrate adhesion and circulatory system process. Conclusion The data show that each endometrial cell-type has a distinct molecular signature and provide a significantly improved overview on the biological process supported by specific cell-types. The most interesting result is that stromal cells express more genes than the two epithelial types and are associated with a greater number of pathways and ontology terms.

scholarly journals Phytochemical Analysis and Potential Biological Activities of Essential Oil from Rice Leaf

Molecules ◽  
2019 ◽  
Vol 24 (3) ◽  
pp. 546 ◽  
Author(s):  
Truong Minh ◽  
Tran Xuan ◽  
Truong Van ◽  
Yusuf Andriana ◽  
Tran Viet ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Essential Oil ◽  
Atmospheric Pressure Chemical Ionization ◽  
Biological Activities ◽  
Methyl Ricinoleate ◽  
Gas Chromatography Mass Spectrometry ◽  
Phytochemical Analysis ◽  
Chemical Compositions ◽  
Rice Leaf ◽  
Rice Leaves

Although many investigations on phytochemicals in rice plant parts and root exudates have been conducted, information on the chemical profile of essential oil (EO) and potent biological activities has been limited. In this study, chemical compositions of rice leaf EO and in vitro biological activities were investigated. From 1.5 kg of fresh rice leaves, an amount of 20 mg EO was obtained by distillation and analyzed by gas chromatography-mass spectrometry (GC-MS), electrospray ionization (ESI), and atmospheric pressure chemical ionization (APCI) to reveal the presence of twelve volatile constituents, of which methyl ricinoleate (27.86%) was the principal compound, followed by palmitic acid (17.34%), and linolenic acid (11.16%), while 2-pentadecanone was the least (2.13%). Two phytoalexin momilactones A and B were first time identified in EO using ultra-performance liquid chromatography coupled with electrospray mass spectrometry (UPLC/ESI-MS) (9.80 and 4.93 ng/g fresh weight, respectively), which accounted for 7.35% and 3.70% of the EO, respectively. The assays of DPPH (IC50 = 73.1 µg/mL), ABTS (IC50 = 198.3 µg/mL), FRAP (IC50 = 700.8 µg/mL) and β-carotene oxidation (LPI = 79%) revealed that EO possessed an excellent antioxidant activity. The xanthine oxidase assay indicated that the anti-hyperuricemia potential was in a moderate level (IC50 = 526 µg/mL) as compared with the standard allopurinol. The EO exerted potent inhibition on growth of Raphanus sativus, Lactuca sativa, and two noxious weeds Echinochloa crus-galli, and Bidens pilosa, but in contrast, the growth of rice seedlings was promoted. Among the examined plants, the growth of the E. crus-galli root was the most inhibited, proposing that constituents found in EO may have potential for the control of the problematic paddy weed E. crus-galli. It was found that the EO of rice leaves contained rich phytochemicals, which were potent in antioxidants and gout treatment, as well as weed management. Findings of this study highlighted the potential value of rice leaves, which may provide extra benefits for rice farmers.

scholarly journals Simultaneous Tracking of Capsid, Tegument, and Envelope Protein Localization in Living Cells Infected with Triply Fluorescent Herpes Simplex Virus 1

2008 ◽  
Vol 82 (11) ◽  
pp. 5198-5211 ◽  
Author(s):  
Ken Sugimoto ◽  
Masashi Uema ◽  
Hiroshi Sagara ◽  
Michiko Tanaka ◽  
Tetsutaro Sata ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Envelope Protein ◽  
Fluorescent Proteins ◽  
Protein Localization ◽  
Envelope Proteins ◽  
Herpes Simplex Virus 1 ◽  
Infected Cells ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT We report here the construction of a triply fluorescent-tagged herpes simplex virus 1 (HSV-1) expressing capsid protein VP26, tegument protein VP22, and envelope protein gB as fusion proteins with monomeric yellow, red, and cyan fluorescent proteins, respectively. The recombinant virus enabled us to monitor the dynamics of these capsid, tegument, and envelope proteins simultaneously in the same live HSV-1-infected cells and to visualize single extracellular virions with three different fluorescent emissions. In Vero cells infected by the triply fluorescent virus, multiple cytoplasmic compartments were found to be induced close to the basal surfaces of the infected cells (the adhesion surfaces of the infected cells on the solid growth substrate). Major capsid, tegument, and envelope proteins accumulated and colocalized in the compartments, as did marker proteins for the trans-Golgi network (TGN) which has been implicated to be the site of HSV-1 secondary envelopment. Moreover, formation of the compartments was correlated with the dynamic redistribution of the TGN proteins induced by HSV-1 infection. These results suggest that HSV-1 infection causes redistribution of TGN membranes to form multiple cytoplasmic compartments, possibly for optimal secondary envelopment. This is the first real evidence for the assembly of all three types of herpesvirus proteins—capsid, tegument, and envelope membrane proteins—in TGN.

scholarly journals Measuring fast gene dynamics in single cells with time-lapse luminescence microscopy

2014 ◽  
Vol 25 (22) ◽  
pp. 3699-3708 ◽  
Author(s):  
Anyimilehidi Mazo-Vargas ◽  
Heungwon Park ◽  
Mert Aydin ◽  
Nicolas E. Buchler
Keyword(s):  
Gene Expression ◽  
Fluorescent Proteins ◽  
Single Cells ◽  
Time Lapse ◽  
Photon Flux ◽  
Luminescence Microscopy ◽  
Cell Cycle Genes ◽  
Light Excitation ◽  
Better Than

Time-lapse fluorescence microscopy is an important tool for measuring in vivo gene dynamics in single cells. However, fluorescent proteins are limited by slow chromophore maturation times and the cellular autofluorescence or phototoxicity that arises from light excitation. An alternative is luciferase, an enzyme that emits photons and is active upon folding. The photon flux per luciferase is significantly lower than that for fluorescent proteins. Thus time-lapse luminescence microscopy has been successfully used to track gene dynamics only in larger organisms and for slower processes, for which more total photons can be collected in one exposure. Here we tested green, yellow, and red beetle luciferases and optimized substrate conditions for in vivo luminescence. By combining time-lapse luminescence microscopy with a microfluidic device, we tracked the dynamics of cell cycle genes in single yeast with subminute exposure times over many generations. Our method was faster and in cells with much smaller volumes than previous work. Fluorescence of an optimized reporter (Venus) lagged luminescence by 15–20 min, which is consistent with its known rate of chromophore maturation in yeast. Our work demonstrates that luciferases are better than fluorescent proteins at faithfully tracking the underlying gene expression.

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