Chloroplasts In Cells Research Articles

Functional annotation of proteins in Catharanthus roseus shoot cultures under biogenic zinc nanotreatment.

Nano-interactions are well known for their positive as well as negative impacts on the morphological and physiological systems of plants. Keeping in mind, the conformational changes in plant proteins as one of the key mechanisms for stress adaptation responses, the current project was designed to explore the effect of glutathione-capped and uncapped zinc nano-entities on Catharanthus roseus shoot cultures. Zinc nanotreatment (0.05μg/mL) significantly induced ester production in C. roseus shoots as detected by Gas Chromatography-Mass spectrometry. These nanotreated shoots were further subjected to peptide-centric nano-LC-MS/MS analysis. Mass spectrometry followed by a Heat map revealed a significant effect of zinc nanoparticles on 59 distinct classes of proteins as compared to control. Proteins involved in regulating stress scavenging, transport, and secondary metabolite biosynthesis were robustly altered under capped zinc nanotreatment. UniProt database identified majority of the localization of the abundantly altered protein in cell membranes and chloroplasts. STRING and Cytoscape analysis assessed inter and intra coordination of triosephosphate isomerase with other identified proteins and highlighted its role in the regulation of protein abundance under applied stress. This study highlights the understanding of complex underlying mechanisms and regulatory networks involved in proteomic alterations and interactions within the plant system to cope with the nano-effect.

Variation of mesophyll conductance mediated by nitrogen form is related to changes in cell wall property and chloroplast number.

Plants primarily incorporate nitrate (NO3 -) and ammonium (NH4 +) as the primary source of inorganic nitrogen (N); the physiological mechanisms of photosynthesis (A) dropdown under NH4 + nutrition has been investigated in many studies. Leaf anatomy is a major determinant to mesophyll conductance (g m) and photosynthesis; however, it remains unclear whether the photosynthesis variations of plants exposed to different N forms is related to leaf anatomical variation. In this work, a common shrub, Lonicera japonica was hydroponically grown under NH4 +, NO3 - and 50% NH4 +/NO3 -. We found that leaf N significantly accumulated under NH4 +, whereas the photosynthesis was significantly decreased, which was mainly caused by a reduced g m. The reduced g m under NH4 + was related to the decreased intercellular air space, the reduced chloroplast number and especially the thicker cell walls. Among the cell wall components, lignin and hemicellulose contents under NH4 + nutrition were significantly higher than those in the other two N forms and were scaled negatively correlated with g m; while pectin content was independent from N forms. Pathway analysis further revealed that the cell wall components might indirectly regulate g m by influencing the thickness of the cell wall. These results highlight the importance of leaf anatomical variation characterized by modifications of chloroplasts number and cell wall thickness and compositions, in the regulation of photosynthesis in response to varied N sources.

Metal toxicity in Bryum coronatum Schwaegrichen: impact on chlorophyll content, lamina cell structure, and metal accumulation

This research examined the impact of heavy metals, including Cd, Pb, and Zn, on chlorophyll content and lamina cell structure in Bryum coronatum. After exposure to varying metal concentrations (0.015, 0.065, 0.250, 1, and 4 mg/L), chlorophyll content, chloroplast numbers, lamina cell change, and metal accumulation were investigated. Chlorophyll content was assessed using spectrophotometry, whereas chloroplast numbers and lamina cell changes were examined under a light microscope. Metal accumulation was quantified through ICP-MS. The findings revealed that Cd notably reduced chlorophyll a content, while Pb and Zn showed minimal influence. Cd and Pb exposure decreased the number of chloroplasts in lamina cells, with no impact from Zn. The moss’s capacity to absorb metals increased with higher exposure levels, indicating its potential as a biomonitor for heavy metal pollution. Cell mortality occurred in response to Cd and Pb, primarily in the median and apical lamina regions, while Zn had no effect. This study sheds light on heavy metal toxicity in B. coronatum, underscoring its significance for environmental monitoring. Further research on the mechanisms and consequences of heavy metal toxicity in bryophytes is essential for a comprehensive understanding of this critical issue.

The C4 photosynthesis bifunctional enzymes, PDRPs, of maize are co-opted to cytoplasmic viral replication complexes to promote infection of a prevalent potyvirus sugarcane mosaic virus.

In maize, two pyruvate orthophosphate dikinase (PPDK) regulatory proteins, ZmPDRP1 and ZmPDRP2, are respectively specific to the chloroplast of mesophyll cells (MCs) and bundle sheath cells (BSCs). Functionally, ZmPDRP1/2 catalyse both phosphorylation/inactivation and dephosphorylation/activation of ZmPPDK, which is implicated as a major rate-limiting enzyme in C4 photosynthesis of maize. Our study here showed that maize plants lacking ZmPDRP1 or silencing of ZmPDRP1/2 confer resistance to a prevalent potyvirus sugarcane mosaic virus (SCMV). We verified that the C-terminal domain (CTD) of ZmPDRP1 plays a key role in promoting viral infection while independent of enzyme activity. Intriguingly, ZmPDRP1 and ZmPDRP2 re-localize to cytoplasmic viral replication complexes (VRCs) following SCMV infection. We identified that SCMV-encoded cytoplasmic inclusions protein CI targets directly ZmPDRP1 or ZmPDRP2 or their CTDs, leading to their re-localization to cytoplasmic VRCs. Moreover, we found that CI could be degraded by the 26S proteasome system, while ZmPDRP1 and ZmPDRP2 could up-regulate the accumulation level of CI through their CTDs by a yet unknown mechanism. Most importantly, with genetic, cell biological and biochemical approaches, we provide evidence that BSCs-specific ZmPDRP2 could accumulate in MCs of Zmpdrp1 knockout (KO) lines, revealing a unique regulatory mechanism crossing different cell types to maintain balanced ZmPPDK phosphorylation, thereby to keep maize normal growth. Together, our findings uncover the genetic link of the two cell-specific maize PDRPs, both of which are co-opted to VRCs to promote viral protein accumulation for robust virus infection.

Uncoupling of stomatal conductance and photosynthesis at high temperatures: mechanistic insights from online stable isotope techniques.

The strong covariation of temperature and vapour pressure deficit (VPD) in nature limits our understanding of the direct effects of temperature on leaf gas exchange. Stable isotopes in CO2 and H2 O vapour provide mechanistic insight into physiological and biochemical processes during leaf gas exchange. We conducted combined leaf gas exchange and online isotope discrimination measurements on four common European tree species across a leaf temperature range of 5-40°C, while maintaining a constant leaf-to-air VPD (0.8 kPa) without soil water limitation. Above the optimum temperature for photosynthesis (30°C) under the controlled environmental conditions, stomatal conductance (gs ) and net photosynthesis rate (An ) decoupled across all tested species, with gs increasing but An decreasing. During this decoupling, mesophyll conductance (cell wall, plasma membrane and chloroplast membrane conductance) consistently and significantly decreased among species; however, this reduction did not lead to reductions in CO2 concentration at the chloroplast surface and stroma. We question the conventional understanding that diffusional limitations of CO2 contribute to the reduction in photosynthesis at high temperatures. We suggest that stomata and mesophyll membranes could work strategically to facilitate transpiration cooling and CO2 supply, thus alleviating heat stress on leaf photosynthetic function, albeit at the cost of reduced water-use efficiency.

Chlamydiae as symbionts of photosynthetic dinoflagellates.

Chlamydiae are ubiquitous intracellular bacteria and infect a wide diversity of eukaryotes, including mammals. However, chlamydiae have never been reported to infect photosynthetic organisms. Here, we describe a novel chlamydial genus and species, Candidatus Algichlamydia australiensis, capable of infecting the photosynthetic dinoflagellate Cladocopium sp. (originally isolated from a scleractinian coral). Algichlamydia australiensis was confirmed to be intracellular by fluorescence in situ hybridization and confocal laser scanning microscopy and temporally stable at the population level by monitoring its relative abundance across four weeks of host growth. Using a combination of short- and long-read sequencing, we recovered a high-quality (completeness 91.73% and contamination 0.27%) metagenome-assembled genome of A. australiensis. Phylogenetic analyses show that this chlamydial taxon represents a new genus and species within the Simkaniaceae family. Algichlamydia australiensis possesses all the hallmark genes for chlamydiae-host interactions, including a complete type III secretion system. In addition, a type IV secretion system is encoded on a plasmid and has previously been observed for only three other chlamydial species. Twenty orthologous groups of genes are unique to A. australiensis, one of which is structurally similar to a protein known from Cyanobacteria and Archaeplastida involved in thylakoid biogenesis and maintenance, hinting at potential chlamydiae interactions with the chloroplasts of Cladocopium cells. Our study shows that chlamydiae infect dinoflagellate symbionts of cnidarians, the first photosynthetic organism reported to harbor chlamydiae, thereby expanding the breadth of chlamydial hosts and providing a new contribution to the discussion around the role of chlamydiae in the establishment of the primary plastid.

Genome-wide identification and characterization of the sucrose invertase gene family in Hemerocallis citrina.

Sucrose invertase is an important catalytic enzyme that is widely distributed in plants and can irreversibly hydrolyze sucrose into fructose and glucose. Daylily is an important perennial flower worldwide and a traditional vegetable in East Asia. Previous studies have suggested that sucrose invertase is involved in the aging of daylily flowers. However, knowledge about the number, physicochemical properties, and expression patterns of daylily sucrose invertases is still lacking. Identifying the daylily sucrose invertase family genes in the genome is highly important for understanding phylogenetic evolution and determining the genetic function of sucrose invertase. To obtain basic knowledge about the number, classification, sequence composition, and physicochemical properties of sucrose invertases in daylily, bioinformatics software was used to analyze the genome of Hemerocallis citrina (H. citrina), and the basic properties of sucrose invertase genes and proteins were obtained. Then, combined with transcriptome data from flower organs at different developmental stages, the expression patterns of each gene were clarified. Finally, the reliability of the transcriptome data was verified by quantitative real-time polymerase chain reaction (PCR). Through software analysis, 35 sucrose invertases were identified from the H. citrina genome and named HcINV1-HcINV35; these enzymes belong to three subfamilies: cell wall invertases, vacuolar invertases, and chloroplast invertases. The amino acid composition, motif types, promoter composition, gene structure, protein physicochemical properties, gene chromosomal localization, and evolutionary adaptability of daylily invertases were determined; these results provided a comprehensive understanding of daylily invertases. The transcriptome expression profile combined with fluorescence quantitative reverse transcription-polymerase chain reaction (RT‒PCR) analysis suggested that almost all daylily invertase genes were expressed in flower organs, but even genes belonging to the same subfamily did not exhibit the same expression pattern at different developmental stages, suggesting that there may be redundancy or dissimilation in the function of daylily sucrose invertases.

Mechanistic transcriptome comprehension of Chlamydomonas reinhardtii subjected to black phosphorus

Two-dimensional materials have recently gained significant awareness. A representative of such materials, black phosphorous (BP), earned attention based on its comprehensive application potential. The presented study focuses on the mode of cellular response underlying the BP interaction with Chlamydomonas reinhardtii as an algal model organism. We observed noticeable ROS formation and changes in outer cellular topology after 72 h of incubation at 5 mg/L BP. Transcriptome profiling was employed to examine C. reinhardtii response after exposure to 25 mg/L BP for a deeper understanding of the associated processes. The RNA sequencing has revealed a comprehensive response with abundant transcript downregulation. The mode of action was attributed to cell wall disruption, ROS elevation, and chloroplast disturbance. Besides many other dysregulated genes, the cell response involved the downregulation of GH9 and gametolysin within a cell wall, pointing to a shift to discrete manipulation with resources. The response also included altered expression of the PRDA1 gene associated with redox governance in chloroplasts implying ROS disharmony. Altered expression of the Cre-miR906–3p, Cre-miR910, and Cre-miR914 pointed to those as potential markers in stress response studies.

Purification of Chloroplast Envelope, Thylakoids, and Stroma from Angiosperm Leaves.

Plant cell chloroplasts are bounded by a two-membrane envelope. Their photosynthetic function is based on the development of an operational large internal membrane network, called the thylakoids, and on enzymatic processes present in the chloroplast matrix, called the stroma. Thylakoid membranes are distinct from the chloroplast envelope, and their biogenesis is dependent on biosynthetic and transport activities specific of the chloroplast envelope. Starting with the isolation of intact chloroplasts, the method presents the separation by differential centrifugation of the three compartments. A protocol is detailed for leaves of spinach, Arabidopsis or pea.

Studies on in vitro chromosome doubling of haploid derived through androgenesis in marigold (Tagetes patula L.)

Local strain of Tagetes patula L. was used for in vitro androgenesis with floret size ranging from 2 to 4.5 mm. One haploid from anther derived regenerants was isolated and confirmed by cytological analysis and chloroplast counting. The isolated haploid was further multiplied and were subjected to colchicine treatment (0.005%, 0.01%, 0.02% for 36 and 48 hours) using nodal segment explants under in vitro conditions. The results revealed that treatment T1- Colchicine (0.005%) for 36 hours exposure was found good for parameters like percent survival (81.22%), percent diploidization (48.00%), leaf length (3.77 cm), leaf width (2.49 cm), number of shoots per plant (9.89) followed by treatment T2- Colchicine (0.005%) for 48 hours exposure for parameters like percent survival (69.67%), percent diploidization (37.22%), leaf length (3.56 cm), leaf width (1.97 cm), number of shoots per plant (8.67). Cytological analysis and chloroplast counting in stomatal guard cells was done for determination of ploidy level in colchicine treated haploid plantlets. The results revealed that in cytological analysis out of 38 plants screened, 26 plants (68.4%) were tetraploids and 12 plants (31.57%) were polyploids. Whereas through chloroplast counting, 38 plants screened out of which 26 plants (68.4%) were tetraploids with mean of 16 chloroplasts in the guard cells and 12 plants (31.57%) were polyploids with mean of 18 to 22 chloroplasts in the guard cells.

Screening of Varieties Resistant to Late-Spring Coldness in Wheat and Effects of Late-Spring Coldness on the Ultrastructure of Wheat Cells

Due to global warming, late-spring coldness affecting wheat (LSCW) is one of the major abiotic adversities affecting wheat production. A combination of field and pot trials were conducted in this study. In the field experiment, 20 wheat varieties from the main wheat-producing areas in China were selected as experimental materials. By exploring the effects of LSCW on the spikelet characteristics and yields of different varieties, the evaluation methods and indexes of wheat varieties’ resistance to LSCW were established. Three varieties with strong resistance to LSCW (Yannong19, Guomai9, and Shannong17) and five varieties sensitive to LSCW (Zhengmai895, Xinmai26, Zhengmai366, Zhengmai8329, and Fanmai5) were screened out. The wheat varieties Yannong19 (YN19), with a strong resistance to LSCW, and Xinmai26 (XM26), with LSCW sensitivity, were selected as the test materials for the pot experiment. The ultrastructure changes in the wheat in different low-temperature treatments during the anther differentiation period were observed using an ultra-low-temperature artificial climate incubator set to 4 °C and −4 °C for 4 h (1:00–5:00 a.m.). The average temperature of the field during the low-temperature treatment was 10 °C, which was the control temperature (CK). The results showed that the cell morphology and chloroplast and mitochondrial structures of the functional leaves, young ears, and internodes below the ears were damaged, and the degree of damage was related to the cell location, the extent of low-temperature stress, and the resistance of the varieties. The degree of damage to the wheat cells was found to occur in the following order: young ears > internode belove young ears > functional leaves. The degrees of damage to the wheat cell, chloroplast, and mitochondrial structures increased with the intensification of the low-temperature stress. The damage to the XM26 variety was obviously greater than that inflicted on the YN19 variety. The anatomical mechanism of YN19 cells makes the cell structure more stable during late-spring coldness.

Stomata variation in the process of polyploidization in Chinese chive (Allium tuberosum).

Stomatal variation, including guard cell (GC) density, size and chloroplast number, is often used to differentiate polyploids from diploids. However, few works have focused on stomatal variation with respect to polyploidization, especially for consecutively different ploidy levels within a plant species. For example, Allium tuberosum, which is mainly a tetraploid (2n = 4x = 32), is also found at other ploidy levels which have not been widely studied yet. We recently found cultivars with different ploidy levels, including those that are diploid (2n = 2x = 16), triploid (2n = 3x = 24), pseudopentaploid (2n = 34-42, mostly 40) and pseudohexaploid (2n = 44-50, mostly 48). GCs were evaluated for their density, size (length and width) and chloroplast number. There was no correspondence between ploidy level and stomatal density, in which anisopolyploids (approximately 57 and 53 stomata/mm2 in triploid and pseudopentaploid, respectively) had a higher stomatal density than isopolyploids (approximately 36, 43, and 44 stomata/mm2 in diploid, tetraploid and pseudohexaploid, respectively). There was a positive relationship between ploidy level and GC chloroplast number (approximately 44, 45, 51, 72 and 90 in diploid to pseudohexaploid, respectively). GC length and width also increased with ploidy level. However, the length increased approximately 1.22 times faster than the width during polyploidization. This study shows that GC size increased with increasing DNA content, but the rate of increase differed between length and width. In the process of polyploidization, plants evolved longer and narrower stomata with more chloroplasts in the GCs.

The cyanobacterial FtsH4 protease controls accumulation of protein factors involved in the biogenesis of photosystem I

Membrane-bound FtsH proteases are universally present in prokaryotes and in mitochondria and chloroplasts of eukaryotic cells. These metalloproteases are often critical for viability and play both protease and chaperone roles to maintain cellular homeostasis. In contrast to most bacteria bearing a single ftsH gene, cyanobacteria typically possess four FtsH proteases (FtsH1–4) forming heteromeric (FtsH1/3 and FtsH2/3) and homomeric (FtsH4) complexes. The functions and substrate repertoire of each complex are however poorly understood. To identify substrates of the FtsH4 protease complex we established a trapping assay in the cyanobacterium Synechocystis PCC 6803 utilizing a proteolytically inactivated trapFtsH4-His. Around 40 proteins were specifically enriched in trapFtsH4 pulldown when compared with the active FtsH4. As the list of putative FtsH4 substrates contained Ycf4 and Ycf37 assembly factors of Photosystem I (PSI), its core PsaB subunit and the IsiA chlorophyll-binding protein that associates with PSI during iron stress, we focused on these PSI-related proteins. Therefore, we analysed their degradation by FtsH4 in vivo in Synechocystis mutants and in vitro using purified substrates. The data confirmed that FtsH4 degrades Ycf4, Ycf37, IsiA, and also the individual PsaA and PsaB subunits in the unassembled state but not when assembled within the PSI complexes. A possible role of FtsH4 in the PSI life-cycle is discussed.

Growth and metabolism of pea (Pisum sativum) via biostimulants based on greener ZnO nanoparticles

The purpose of this study was to identify the most important physiological and biological effects of green synthesis ZnO nanoparticles at a size of 65 nm, biostimulant (Folcare) and interaction biostimulant ZnO NPs on plant growth and metabolism. As our understanding of biostimulants’ preventive and restorative modes of action has increased, it is critical to maintain the best crop output and quality possible. The reduction of fertilizers must be substituted by strategies that improve the nutrients uptake or their utilization by the plants. New processing methods are required as an efficient green process or an integrated (hybrid) process for different new technologies of interest. The effects of NPs, biostimulant, and combination ZnO NPs biostimulant on plant cell metabolism were examined in cytosol, chloroplast, and mitochondria of cells from the stems, roots, and leaves. The interaction NPs/biostimulant had a beneficial effect on the morphological and physiological indicators of plant health than when nanoparticles and biostimulant are applied separately. Folcare biostimulant coupled with zinc oxide nanoparticles improved pea crops growth. The improved of the quality of pea plants can be explained at least, in part, by increase in antioxidant activities during plant growth phenophase.

Engineering of insecticidal hybrid gene into potato chloroplast genome exhibits promising control of Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae).

The potato chloroplast was transformed with codon optimized synthetic hybrid cry gene (SN19) to mitigate crop losses by Colorado potato beetle (CPB). The bombarded explants (leaves and internode) were cultured on MS medium supplemented with BAP (2.0mg/l), NAA (0.2mg/l), TDZ (2.0mg/l) and GA3 (0.1mg/l); spectinomycin 50mg/l was used as a selection agent in the medium. Leaf explants of cultivar Kuroda induced highest percentage (92%) of callus where cultivar Santae produced the highest percentage (85.7%) of transplastomic shoots. Sante and Challenger showed 9.6% shoot regeneration efficiency followed by cultivar Simply Red (8.8%). PCR amplification yielded 16 postive transplastomic plantlets out of 21 spectinomycin resistant ones. Target gene integration was confirmed by PCR and Southern blot, whereas RT-qPCR was used to assess the expression level of transgene. The localization of visual marker gene gfp was tracked by laser scanning confocal microscopy which confirmed its expression in chloroplasts of leaf cells. The transplastomic plants ensured high mortality to both larvae and adult CPB. Foliage consumption and weight gain of CPB fed on transplastomic leaves were lower compared to the control plants. Sucessful implementation of current research findings can lead to a viable solution to CPB mediated potato losses globally.

Mitochondrial DNA: From Forensic Genetics to the Origins of Humanity

Mitochondria are cellular organelles that provide cellular energy. Along with chloroplasts of plant cells, mitochondria are the only organelles that have genetic material. These peculiarities make mitochondrial DNA (DNA) one of the main research topics in the field of Cytology and Genetics. Interest shared by Criminalistics, which uses mDNA analysis for forensic identification of DNA traces in which nuclear DNA analysis is not possible. In addition to being used in forensic investigation, mtDNA analysis has also been widely used in paleoanthropological investigation through the elaboration of human phylogenies, which retroact to only one female ancestor, known as mitochondrial Eve. In this context, this manuscript aims to address the relevance of this organelle in the field of Criminalistics and the Sciences of Origins, presenting the scientific, philosophical and theological implications arising from the in-depth analysis of this cellular structure.

Ultrastructural organization of the thylakoid system during the afternoon relocation of the giant chloroplast in Selaginella martensii Spring (Lycopodiophyta)

Within the ancient vascular plant lineage known as lycophytes, many Selaginella species contain only one giant chloroplast in the upper epidermal cells of the leaf. In deep-shade species, such as S. martensii, the chloroplast is cup-shaped and the thylakoid system differentiates into an upper lamellar region and a lower granal region (bizonoplast). In this report, we describe the ultrastructural changes occurring in the giant chloroplast hosted in the epidermal cells of S. martensii during the daily relocation of the organelle. The process occurs in up to ca. 40% of the microphylls without the plants being exposed to high-light flecks. The relocated chloroplast loses its cup shape: first, it flattens laterally toward the radial cell wall and then assumes a more globular shape. The loss of the conical cell shape, the side-by-side lateral positioning of vacuole and chloroplast, and the extensive rearrangement of the thylakoid system to only granal cooperate in limiting light absorption. While the cup-shaped chloroplast emphasizes the light-harvesting capacity in the morning, the relocated chloroplast is suggested to support the renewal of the thylakoid system during the afternoon, including the recovery of photosystem II (PSII) from photoinhibition. The giant chloroplast repositioning is part of a complex reversible reshaping of the whole epidermal cell.

Intracellular positioning of mesophyll chloroplasts following to aggregative movement in Setaria viridis analysed three-dimensionally with a confocal laser scanning microscope

Chloroplasts can change their intracellular position responding to environmental conditions. In addition to the well-known photorelocation movements, i.e., accumulation and avoidance movements in response to light, mesophyll chloroplasts in C4 plants change their intracellular positioning from the cell periphery to the bundle sheath cell side (aggregative movement) in response to environmental stresses such as drought, salinity, and hyperosmosis, under light. Previous studies on the aggregative movement have been limited in two-dimensional (2D) information on the transverse sections of leaves, because aggregated mesophyll chloroplasts gather inside the leaf tissues, which need to be cut for observation. However, the 2D analysis on the cross sections is difficult to investigate accurately the aggregated chloroplasts overlapped each other in the depth direction. Therefore, there are few studies examining the anatomical features of the aggregated chloroplasts at the cellular level. Here in this study, we established the workflow for three-dimensional (3D) observation using a confocal laser scanning microscope (CLSM), which can investigate a thick section as a stack of optical sections, followed by 3D reconstruction of mesophyll cells and chloroplasts. Using this method, we visualized the 3D representations of mesophyll cells of green foxtail (Setaria viridis), which is a model of C4 plant, and investigated the chloroplasts individually and quantified their structures or intracellular positions before and after the aggregative movement. The 3D data of individual chloroplasts in a whole cell revealed that the aggregated positioning is independent with chloroplast volume or surface area, and that chloroplasts did not change their shape before and after the movement.

Biocontrol of chocolate spot disease of broad bean (Vicia faba L.) caused by Botrytis fabae using biosynthesized reduced graphene oxide/silver nanocomposite

This study was demonstrated to investigate the effects of reduced graphene oxide/silver nanocomposite (rGO/AgNC) on controlling the chocolate spot disease of broad bean (Vicia faba L.) caused by Botrytis fabae. rGO/AgNC was green synthesized using the cell-free supernatant of Escherichia coli D8 in the presence of sunlight and room temperature. rGO/AgNC revealed a broad UV–vis absorption peak at 433 nm. Transmission electron microscopy (TEM) images of rGO/AgNC confirmed the presence of spherical-shaped silver nanoparticles (AgNPs) with an average size of 7–26 nm. Zeta analysis of rGO/AgNC displayed their positive charge (+18.9 mV) and the good dispersion of AgNPs on the surface of rGO. rGO/AgNC had good antifungal activity against B. fabae in a dose-dependent pattern. In vivo, rGO/AgNC-treatment study on faba bean plants showed the biocidal action of rGO/AgNC (150 μg/ml) against B. fabae and completely prevented the pathogen infection. In addition, there was an increase in total phenolic content, peroxidase, and polyphenol oxidase activity, and protein content of rGO/AgNC-treated plants. Plants treated with rGO/AgNC displayed a typical cell morphology in their ultrastructure, including cell membranes and chloroplasts with ellipsoidal shapes and large starch grains. These are all encouraging findings for the use of the biosynthesized rGO/AgNC as a safe and efficient antifungal agent against B. fabae.

A rapid and robust leaf ablation method to visualize bundle sheath cells and chloroplasts in C3 and C4 grasses

BackgroundIt has been proposed that engineering the C4 photosynthetic pathway into C3 crops could significantly increase yield. This goal requires an increase in the chloroplast compartment of bundle sheath cells in C3 species. To facilitate large-scale testing of candidate regulators of chloroplast development in the rice bundle sheath, a simple and robust method to phenotype this tissue in C3 species is required.ResultsWe established a leaf ablation method to accelerate phenotyping of rice bundle sheath cells. The bundle sheath cells and chloroplasts were visualized using light and confocal laser microscopy. Bundle sheath cell dimensions, chloroplast area and chloroplast number per cell were measured from the images obtained by confocal laser microscopy. Bundle sheath cell dimensions of maize were also measured and compared with rice. Our data show that bundle sheath width but not length significantly differed between C3 rice and C4 maize. Comparison of paradermal versus transverse bundle sheath cell width indicated that bundle sheath cells were intact after leaf ablation. Moreover, comparisons of planar chloroplast areas and chloroplast numbers per bundle sheath cell between wild-type and transgenic rice lines expressing the maize GOLDEN-2 (ZmG2) showed that the leaf ablation method allowed differences in chloroplast parameters to be detected.ConclusionsLeaf ablation is a simple approach to accessing bundle sheath cell files in C3 species. We show that this method is suitable for obtaining parameters associated with bundle sheath cell size, chloroplast area and chloroplast number per cell.