Marker Lines Research Articles

Bright Fluorescent Vacuolar Marker Lines Allow Vacuolar Tracing Across Multiple Tissues and Stress Conditions in Rice

The vacuole is indispensable for cells to maintain their water potential and to respond to environmental changes. Nevertheless, investigations of vacuole morphology and its functions have been limited to Arabidopsis thaliana with few studies in the model crop rice (Oryza sativa). Here, we report the establishment of bright rice vacuole fluorescent reporter systems using OsTIP1;1, a tonoplast water channel protein, fused to either an enhanced green fluorescent protein or an mCherry red fluorescent protein. We used the corresponding transgenic rice lines to trace the vacuole morphology in roots, leaves, anthers, and pollen grains. Notably, we observed dynamic changes in vacuole morphologies in pollen and root epidermis that corresponded to their developmental states as well as vacuole shape alterations in response to abiotic stresses. Our results indicate that the application of our vacuole markers may aid in understanding rice vacuole function and structure across different tissues and environmental conditions in rice.

Root-knot nematodes induce gall formation by recruiting developmental pathways of post-embryonic organogenesis and regeneration to promote transient pluripotency.

Root-knot nematodes (RKNs; Meloidogyne spp.) induce new post-embryogenic organs within the roots (galls) where they stablish and differentiate nematode feeding cells, giant cells (GCs). The developmental programmes and functional genes involved remain poorly defined. Arabidopsis root apical meristem (RAM), lateral root (LR) and callus marker lines, SHORT-ROOT/SHR, SCARECROW/SCR, SCHIZORIZA/SCZ, WUSCHEL-RELATED-HOMEOBOX-5/WOX5, AUXIN-RESPONSIVE-FACTOR-5/ARF5, ARABIDOPSIS-HISTIDINE PHOSPHOTRANSFER-PROTEIN-6/AHP6, GATA-TRANSCRIPTION FACTOR-23/GATA23 and S-PHASE-KINASE-ASSOCIATED-PROTEIN2B/SKP2B, were analysed for nematode-dependent expression. Their corresponding loss-of-function lines, including those for LR upstream regulators, SOLITARY ROOT/SLR/IAA14, BONDELOS/BDL/IAA12 and INDOLE-3-ACETIC-ACID-INDUCIBLE-28/IAA28, were tested for RKN resistance/tolerance. LR genes, for example ARF5 (key factor for root stem-cell niche regeneration), GATA23 (which specifies pluripotent founder cells) and AHP6 (cytokinin-signalling-inhibitor regulating pericycle cell-divisions orientation), show a crucial function during gall formation. RKNs do not compromise the number of founder cells or LR primordia but locally induce gall formation possibly by tuning the auxin/cytokinin balance in which AHP6 might be necessary. Key RAM marker genes were induced and functional in galls. Therefore, the activation of plant developmental programmes promoting transient-pluripotency/stemness leads to the generation of quiescent-centre and meristematic-like cell identities within the vascular cylinder of galls. Nematodes enlist developmental pathways of new organogenesis and/or root regeneration in the vascular cells of galls. This should determine meristematic cell identities with sufficient transient pluripotency for gall organogenesis.

Simple Timing Signal Analysis Device-XY Mode Displays the Multichannel Shift Waveform

We designed and implemented a simple timing signal analysis device, which displayed multichannel shift waveform without overlapping from left to right on the Oscilloscope screen using X-Y mode as signal input. In order to meet the requirements of high speed sampling rate, we took microcontroller STM32F407VGT6 to collect, storage, analysis signal, and calculate the result wave though high speed DA conversion module to display on the Oscilloscope screen. we took microcontroller STM32F103RB to realize the shift clock circuit and 8-channel digital signal, and adopt the dial-code switch presents the 8-bit trigger word TW. after several test, the signal analysis device could realize the trigger time position adjust at all, time marker line be shifted manually, LED light displayed the status word corresponding to the flag line, and playback two shift cycle timing waveform and other functions simply and efficient. through numerical simulation and experimental analysis, we found that this simple device can greatly display multichannel shift waveform easily and precisely.

Titanium Dioxide Nanoparticles Promote Root Growth by Interfering with Auxin Pathways in Arabidopsis thaliana

TiO2 nanoparticles (nano-TiO2) are widely used in the world, and a considerable amount of nano-TiO2 is released into the environment, with toxic effects on organisms. In the various species of higher plants, growth, including seed germination, root elongation, and biomass accumulation, is affected by nano-TiO2. However, the underlying molecular mechanisms remain to be elucidated. In this study, we observed that nano-TiO2 promoted root elongation in a dose-dependent manner. Furthermore, we found that nano-TiO2 elevated auxin accumulation in the root tips of the auxin marker lines DII-VENUS and DR5:: GUS, and, correspondingly, quantitative real-time PCR analysis revealed that nano-TiO2 increased the expression levels of auxin biosynthesis- and transportrelated genes. GFP fluorescence observation using transgenic PIN2-GFP indicated that nano-TiO2 promoted root growth by inducing PIN2 accumulation. Thus, we propose that nano-TiO2 promote root growth in Arabidopsis thaliana by altering the expression levels of auxin biosynthesis- and transport-related genes.

变光照环境下车道标识线的识别方法

变光照环境下车道标识线的识别方法

Imaging of Embryo Sac and Early Seed Development in Maize after Feulgen Staining.

Compared with small model plants like Arabidopsis containing ovules with few cell layers, embryo sac and embryo development of model crop plants such as maize and other grasses are difficult to image. Multiple layers of tissue usually surround the deeply embedded embryo sac and developing embryo. Moreover, reliable cell biological marker lines labeling, for example, nuclei, plasma membrane, cell walls, or cells of a specific identity are often not available. The introduction of markers to study mutants is difficult and time-consuming and may require several generations of backcrosses. In this chapter, we therefore present an easy protocol to image maize ovaries and developing embryo sacs before and after fertilization allowing also high-throughput mutant analysis. The laborious embedding of samples and preparation of thin sections are omitted in this fixing-Feulgen staining-clearing (FFC) method. Optical sectioning through multiple layers of tissue is possible allowing 3D reconstructions of the whole embryo sac if necessary. The advantage of staining cell nuclei using the FFC method described here compared, for example, with DAPI staining is a wide range of Schiff's type reagents available for the Feulgen reaction. Depending on the reagent of choice, various conditions such as different excitation/emission filters or even white light can be applied for imaging. Moreover, in order to better visualize cell division, nuclei polarity as well as cell extent and integrity, periodic acid staining (PAS) of cell walls can be combined with Feulgen staining.

The influence of genes which responsible for leaf shape and lines height on the economic qualities of Helianthus annuus hybrids

Purpose. To study the influence of the Dw, Fr, sp genes which control the morphological markers of lines on the economically valuable indicators of the first-generation hybrids.Methods. Field experiment, morphological description and biometric measurements, statistical analysis.Results. The analogue lines were crossed by genes responsible for morphological characteristics of the leaf shape sp (spoon-shaped), leaf edge Fr (fringe), stem Dw (dwarf) and the initial selection lines with sterile lines. The cross-bred hybrids were evaluated in the field experiment on the manifestation of marker trait, yield, oil content, weight of 1000 seeds, height, diameter of the basket (head), the number of leaves. Indicators of line hybrids with morphological marker lines were compared with indicators of hybrids with baseline lines without marker features. The recessive allele of the sp gene determines the spoon-like shape of the leaf, with edges bend upward. This sing is not observed in the first-generation hybrids. Fr gene in the dominant state determines the strongly rugged edge of the leaf. In hybrids of the first generation, changes in the edge of the leaf plate are also observed. The third sign - dwarf of sunflower is determined by the Dw gene, which in the dominant homozygous state reduces the height of plants to 90–100 cm. In the heterozygous state, plants also have a decrease in height. Three breeding lines were used to create analogue lines according to these three criteria. There were two parent ‘LVO7V’, ‘ZL678V’ and one maternal ‘L06B’. Six maternal sterile lines were used to cross with analogue lines. Hybrids were studied by yield, oil content, weight of 1000 seeds, plant height, head diameter, number of leaves.Conclusions. A high probability of the negative impact of the dominant allele of the Dw gene (dwarfishness – short stem height) on the hybrids productivity was found. The high probability of a positive effect of the recessive allele of the sp gene (spoon shaped leaf) on the yield, oil content and number of leaves of hybrids with a line carrier of recessive homozygotes has been proved. The moderate and low probabilities of the difference in the level of signs of hybrids with the dominant allele of the Fr gene (fringe of the leaf edge) and hybrids with baseline lines were determined.

Green Fluorescent Protein- and Discosoma sp. Red Fluorescent Protein-Tagged Organelle Marker Lines for Protein Subcellular Localization in Rice.

The subcellular localization of proteins is a fundamental aspect of protein functions. Determining the subcellular localization is important for understanding the biological functions of proteins. Here, we developed a set of rice organelle marker lines, in which the expressing fluorescent organelle markers could be used as comparative standards in determining the subcellular localization of the protein of interest. We constructed green fluorescent protein (GFP)- and/or Discosoma sp. red fluorescent protein (DsRed)-tagged organelle markers targeted to the endoplasmic reticulum (ER), mitochondria, Golgi apparatus, peroxisome, actin cytoskeleton, plastid, tonoplast, plasma membrane, and nucleus, respectively. The utility of the rice marker lines for protein subcellular localization studies was demonstrated by detecting a nucleus-localized OsWRKY45 and a mitochondria-associated NbHxk1 in protoplasts of the GFP-OsH2B and the ScCOX4-DsRed lines, respectively. Using a sheath-inoculation method, followed by a live-cell imaging, we detected co-localization of a Magnaporthe oryzae PWL2:mCherry : NLS fusion with the nucleus marker in the GFP-OsH2B rice epidermal cells, confirming the translocation of the M. oryzae effector PWL2 into host cells, and further demonstrating the feasibility of using the organelle marker lines for studying dynamics of proteins in rice cells in the interactions between rice and pathogens. The set of organelle marker lines developed in the present study, provides a valuable resource for protein subcellular localization studies in rice.

Application of three-dimensional reconstruction to improve the preoperative measurement accuracy and applicability of femoral neck torsion angle.

Conventional methods have limitations in measuring femoral neck torsion angle (FNTA) of patients with femoral deformities. A new method of three-dimensional (3D) reconstruction technology based on computer tomography (CT) was proposed to enhance measurement accuracy and applicability in this study.Bilateral FNTA of 50 developmental dysplasia of the hip (DDH) patients (DDH group) and 81 volunteers (normal group) were measured by Mimics software based on CT data with the marker lines determined by centerline and curvature. Each FNTA was measured by observer A and observer B for twice separately. 50 DDH patients were classified into 3 groups (group I, II, III) according to Hartofilakidis classification. The statistical analysis of the differences was made among the measurements of the FNTA.The FNTA values were 27.56° ± 12.48° in DDH group and 21.22° ± 8.14° in normal group with significant difference (t = 4.516, P < .001). The FNTA values were 24.53° ± 2.40° in group I, 29.78° ± 1.83° in group II and 39.08° ± 3.13° in group III, with significant difference (F = 7.568, P = .001).The accuracy, reliability and applicable scope of FNTA measurement can be improved by 3D reconstruction in clinical practice. The applicable scope of this method included normal people and patients with femoral deformities. The FNTA of DDH patients is significantly larger than normal volunteers with a positive correlation between the severity of classification. This study will also provide references for preoperative design of Chinese population.

Learning‐based method for lane detection using regionlet representation

Lane detection is an important enabling or enhancing technology for many intelligent applications. A marker line can be segmented into several image blocks, each of which contains lane marking in the centre. This study proposes a learning-based method for lane localisation via detecting and grouping such image blocks. The authors model the marking class using regionlet representation, in which each image block is regarded as a region and is represented by a group of regionlets. A region feature composed of the features extracted from the regionlets contributes a weak classifier. A cascade structure detector is then trained for lane detection. At early stages, it rejects as many negatives as possible. Each layer of the cascade detector is a strong classifier, which consists of several weak classifiers. A real AdaBoost algorithm is adopted to select the most discriminative features and to train the classifiers. Since the use of regionlet features allows desired performance with only a few weak classifiers and the dimensionality of the features is significantly reduced by principal component analysis, the computational burden of their algorithm is much lower than other learning-based methods. Experiment results demonstrate the computational efficiency and robustness of the method.

Phosphoethanolamine N-methyltransferase 1 contributes to maintenance of root apical meristem by affecting ROS and auxin-regulated cell differentiation in Arabidopsis.

The continuous growth of roots requires the balance between cell division and differentiation. Reactive oxygen species (ROS) and auxin are important regulators of root development by affecting cell division and differentiation. The mechanism controlling the coordination of cell division and differentiation is not well understood. Using a forward genetic screen, we isolated a mutant, defective primary root 2 (dpr2), defective in root apical meristem (RAM) maintenance. The DPR2 gene encodes phosphoethanolamine N-methyltransferase 1 (PEAMT1) that catalyzes phosphocholine biosynthesis in Arabidopsis. We characterized the primary root phenotypes of dpr2 using various marker lines, using histochemical and pharmacological analysis to probe early root development. Loss-of-function of DPR2/PEAMT1 resulted in RAM consumption by affecting root stem cell niche, division zone, elongation and differentiation zone (EDZ). PIN-FORMED (PIN) protein abundance, PIN2 polar distribution and general endocytosis were impaired in the root tip of dpr2. Excess hydrogen peroxide and auxin accumulate in the EDZ of dpr2, leading to RAM consumption by accelerating cell differentiation. Suppression of ROS over-accumulation or inhibition of auxin signalling partially prevent RAM differentiation in dpr2 after choline starvation. Taken together, we conclude that the EDZ of the root tip is most sensitive to choline shortage, leading to RAM consumption through an ROS-auxin regulation module.

Cover Image: Cortical microtubule arrays in trichomes

Microtubules play pivotal roles in multifaceted fundamental cellular processes. In this issue, Liu et al. (388–393) report a series of novel microtubule fluorescent marker lines for better recording microtubule architectures and dynamics in living plant cells. These marker lines produce bright, stable fluorescent signals in various tissues.

A Simple, Inexpensive, and Precise Photographic Method for Intraoperative Toric IOL Alignment.

It was the aim of this study to analyse the refractive outcome of toric intraocular lens (t-IOL) implantation guided by a newly developed method of photographic alignment. Fourteen eyes of 10 patients (6 females, 4 males, age 63.4 ± 6.7 (40.6 - 68.0) years [mean ± 1 SD (range)]) were included in this retrospective study. All eyes received an Oculentis Tplus LS-313 (Oculentis, Berlin, Germany) t-IOL after standard phacoemusification (n = 4) or femtocataract laser-assisted surgery (n = 10). Image-guided t-IOL alignment included: (1) calculation of t-IOL parameters (combination of different biometric methods) and plotting a layout drawing of the target lens position, (2) capture of an anterior segment slit lamp photograph in the upright position, (3) superposition of the photographs by the t-IOL target plot and a gradual scale, (4) drawing of marker lines on the photograph and removal of the IOL target coordinates, (5) transfer of the final image to a tablet PC, and (6) marking of the target axis at the corneal limbus with an ink pen in the surgical room. Preoperative corneal astigmatism was 2.50 ± 0.97 (1.38 - 4.34) diopters (D) (mean ± 1 SD, range). In all eyes, intraoperative alignment could easily be performed using the photographic target layout. Surgical interventions and postoperative follow-up were without complications in all cases. Residual postoperative astigmatism was 0.16 ± 0.24 (0.00 - 0.75) D (p < 0.001 compared to preoperative astigmatism). In 64% of patients, residual subjective astigmatism was zero, in 93% ≤ 0.5 D, and in 100% ≤ 0.75 D. Postoperative uncorrected logMAR visual acuity (0.05 ± 0.07, range 0.00 - 0.20) and best-corrected visual acuity (0.03 ± 0.05, 0.00 - 0.10) were significantly better (each p < 0.001) than best-corrected preoperative visual acuity (0.21 ± 0.14, 0.00 - 0.49). Results of this clinical series clearly indicate that the newly developed photographically guided technique of toric lens alignment leads to highly accurate postoperative results for astigmatic correction.

Hybridization and rapid differentiation after secondary contact between the native green anole (Anolis carolinensis) and the introduced green anole (Anolis porcatus).

In allopatric species, reproductive isolation evolves through the accumulation of genetic incompatibilities. The degree of divergence required for complete reproductive isolation is highly variable across taxa, which makes the outcome of secondary contact between allopatric species unpredictable. Since before the Pliocene, two species of Anolis lizards, Anolis carolinensis and Anolis porcatus, have been allopatric, yet this period of independent evolution has not led to substantial species‐specific morphological differentiation, and therefore, they might not be reproductively isolated. In this study, we determined the genetic consequences of localized, secondary contact between the native green anole, A. carolinensis, and the introduced Cuban green anole, A. porcatus, in South Miami. Using 18 microsatellite markers, we found that the South Miami population formed a genetic cluster distinct from both parental species. Mitochondrial DNA revealed maternal A. porcatus ancestry for 35% of the individuals sampled from this population, indicating a high degree of cytonuclear discordance. Thus, hybridization with A. porcatus, not just population structure within A. carolinensis, may be responsible for the genetic distinctiveness of this population. Using tree‐based maximum‐likelihood analysis, we found support for a more recent, secondary introduction of A. porcatus to Florida. Evidence that ~33% of the nuclear DNA resulted from a secondary introduction supports the hybrid origin of the green anole population in South Miami. We used multiple lines of evidence and multiple genetic markers to reconstruct otherwise cryptic patterns of species introduction and hybridization. Genetic evidence for a lack of reproductive isolation, as well as morphological similarities between the two species, supports revising the taxonomy of A. carolinensis to include A. porcatus from western Cuba. Future studies should target the current geographic extent of introgression originating from the past injection of genetic material from Cuban green anoles and determine the consequences for the evolutionary trajectory of green anole populations in southern Florida.

Live-Cell Imaging of Fluorescently Tagged Phloem Proteins with Confocal Microscopy.

Confocal laser scanning microscopy can enable observation of phloem cells in living tissues. Here we describe live imaging of phloem cells in the leaves and roots of Arabidopsis thaliana using fluorescently tagged proteins, either expressed in the vasculature using phloem specific promoters or constitutively expressed reference marker lines. Now, the majority of phloem cell types can be identified, allowing a precise cellular and subcellular localization of phloem proteins.

CLASP Facilitates Transitions between Cortical Microtubule Array Patterns.

Acentrosomal plant microtubule arrays form patterns at the cell cortex that influence cellular morphogenesis by templating the deposition of cell wall materials, but the molecular basis by which the microtubules form the cortical array patterns remains largely unknown. Loss of the Arabidopsis (Arabidopsis thaliana) microtubule-associated protein, CYTOPLASMIC LINKER ASSOCIATED PROTEIN (AtCLASP), results in cellular growth anisotropy defects in hypocotyl cells. We examined the microtubule array patterning in atclasp-1 null mutants and discovered a significant defect in the timing of transitions between array patterns but no substantive defect in the array patterns per se. Detailed analysis and computational modeling of the microtubule dynamics in two atclasp-1 fluorescent tubulin marker lines revealed marker-dependent effects on depolymerization and catastrophe frequency predicted to alter the steady-state microtubule population. Quantitative in vivo analysis of the underlying microtubule array architecture showed that AtCLASP is required to maintain the number of growing microtubule plus ends during transitions between array patterns. We propose that AtCLASP plays a critical role in cellular morphogenesis through actions on new microtubules that facilitate array transitions.

Implementation of autonomous driving of a ground vehicle for narrow high-curvature roads using surround view images

In this paper, a new approach for the implementation of autonomous driving of a ground vehicle on narrow and high-curvature roads using a surround view system is proposed. The approach called the independent frame consecutive searching technique is an algorithm which is developed to detect the left and right lane markers of a lane stably and to generate virtual lane marker lines especially when the roads are narrow and have sudden corners and windings with some faded lane markers. Then the steering angle of an autonomous vehicle is calculated so that the vehicle drives along the desired path after deriving the smoothed virtual centerline. In the real-time experiment, the suggested approach achieved robust detection of lane markers using surround view images and drove a test vehicle autonomously with the generated desired paths on such narrow and high-curvature roads with any lane departures. Therefore, this study shows that the method can be applied to the development of autonomous vehicles.

Simple Culture Methods and Treatment to Study Hormonal Regulation of Ovule Development.

Ovule development is one of the most important processes in the reproductive development of higher plants and is a determinant of seed quality and quantity. Phytohormones play key roles in this process since loss-of-function mutants in hormone signaling show defective ovule phenotypes and reduced fertility. However, it is difficult to distinguish the direct effects of hormones on ovule development because it is parts of reproductive development and the defective phenotypes would be the indirect effects following the defective vegetative development. The treatment of hormones is a direct method to investigate the hormonal regulation of ovule development, but ovule is embedded inside several layers of floral organs, and traditional methods for hormone (or inhibitor) treatments have various limitations. We have developed simple methods to apply treatments to the flowers in a living plant, where an inflorescence apex is immersed into a solution in an inverted tube. We have also developed a specific system to culture and treat excised flowers/pistils. These procedures will be useful for research on the hormonal regulation of ovule development. We provide examples of how treatments with brassinosteroids (BR) and BR biosynthesis inhibitor. We cultured and treated plant materials using our newly developed methods, and observed the morphology of wild type ovules and fluorescence signals in a marker line to monitor the progress of ovule development. The results demonstrate BR promotes ovule development and our new methods are efficient and repeatable.

The role of podocytes dysfunction in chronic glomerulonephritis progression.

In the review, the mechanisms of podocytes damage underlying the development of proteinuria and progression of glomerulosclerosis in chronic glomerulonephritis are discussed in detail. The results of experimental and clinical studies are presented. Under the different immune and non-immune factors the podocytes form a stereotyped response to damage consisting in the reorganization of the actin cytoskeleton, foot process effacement, the detachment of podocytes from the glomerular basement membrane, and the appearance of specific podocyte proteins and whole cells (podocyturia) in the urine. Massive podocyturia in a limited proliferative capacity of podocytes leads to reduce their total count in the glomerulus (podocytopenia) and the development of glomerulosclerosis. The authors describe the line of markers of the podocyte injury and invasive and non-invasive methods of their assessment. In addition, the relationship of podocyturia level with proteinuria and renal dysfunction are discussed, the prospects of assessment the podocyte proteins in urine for assessing of glomerular damage severity and glomerulosclerosis risk are examined.

Identification of a specific molecular marker for the rice blast-resistant gene Pigm and molecular breeding of thermo-sensitive genic male sterile leaf-color marker lines

Rice blast is a damaging disease caused by Magnaportheoryzae. Marker-assisted selection of blast resistance genes could help develop cultivars with blast resistance. Pigm is a broad-spectrum blast-resistant gene. However, few rice resources contain Pigm. In this study, the Pigm gene donor Gumei4 (GM4) was investigated. By analyzing different regions of Pigm sequences, we found that marker G8900 was a specific molecular marker of Pigm gene in GM4. Correlation analysis between molecular marker detection and identification of rice blast disease nursery revealed that G8900 could be used in marker-assisted selection (MAS) of Pigm. Furthermore, we introduced Pigm gene into the KT27S line (a blast-susceptible yellow-green-leaf-color mutant) in G8900-assisted breeding and identified three new yellow-green-leaf-color marker lines that are resistant to blast. The agronomic and economic traits of the three new lines are similar to those of their parental lines. The identification and application of Pigm-specific molecular marker in breeding of yellow-green-leaf-color marker line could play an important role in the production of disease-resistant hybrid rice.