A frozen suspension external calibration strategy for elemental quantitative imaging of fresh plant soft tissues by LA-ICP-MS with cryogenic ablation cell.

A simple and reliable external calibration strategy of LA-ICP-MS for fresh plant soft tissues was developed. The prepared plant suspension was frozen by the designed cryogenic ablation cell and used as external standard for quantitative elemental imaging analysis of fresh plant tissues. The controllable water content of the prepared external standards provides a similar matrix with fresh soft tissues, and a homogeneous elemental distribution could be ensured due to the fine grinding particle sizes. More interestingly, the presence of water increased the signal intensity produced by the suspension by a factor of 1.6 (Pb) to 66.6 (La) compared to that of the pressed cake. The excellent dispersing property and advantage of long-term use were achieved owing to the employment of 0.1% PAANa as suspending agent. A series of plant reference materials were analyzed, and the relative errors of most elements were less than 10 %, indicating that there is a reliable accuracy of the proposed method. The limits of detection (LODs) ranged from 0.1 ng·g-1 (La) to 1279 ng·g-1 (S). This method was used for elemental imaging analysis in rice leaves under arsenic stress, and the results were consistent with previous studies, which mean that the proposed method could provide technical support for researchers in the fields of agriculture and environment.

Immunofluorescence staining of chloroplast proteins with frozen sections of plant tissues.

KEY MESSAGE: Immunofluorescence staining with frozen sections of plant tissues and a nest tube is convenient and effective, and broadens the applicability of immunofluorescence staining. Immunofluorescence staining is an indispensable and extensively employed technique for determining the subcellular localization of chloroplast division proteins. At present, it is difficult to effectively observe the localization of target proteins in leaves that are hard, or very thin, or have epidermal hair or glands with the current immunofluorescence staining methods. Moreover, signals of target proteins were predominantly detected in mesophyll cells, not the cells of other types. Thus, the method of immunofluorescence staining was further explored for improvement in this study. The plant tissue was embedded with 50% PEG4000 at -60℃, which was then cut into sections by a cryomacrotome. The sections were immediately immersed in fixation solution. Then, the sample was transferred into a special nested plastic tube, which facilitated the fixation and immunofluorescence staining procedures. The use of frozen sections in this method enabled a short processing time and reduced material requirements. By optimizing the thickness of the sections, a large proportion of the cells could be well stained. With this method, we observed the localization of a chloroplast division protein FtsZ1 in the wild-type Arabidopsis and various chloroplast division mutants. Meanwhile, the localization of FtsZ1 was also observed not only in mesophyll cells, but also in guard cells and epidermal cells in a lot of other plant species, including many species with hard leaf tissues. This method is not only easy to use, but also expands the scope of applicability for immunofluorescence staining.

Bioaccumulation and distribution of Pb, Ni, Zn and Fe in stinging nettle (Urtica dioica) tissues and heavy metal-contamination assessment in the industrial zone of smelter Ferronikeli (Drenas-Kosovo).

Here, we determined the concentrations of Pb, Ni, Zn and Fe in the soil and in vegetative organs of stinging nettle (Urtica dioica) collected from the banks of the Drenica River in the vicinity of the Ferronikeli smelter. The results were compared with samples collected from the banks 20 km (Shalë village) upriver. In addition, the bioaccumulation factor (BCF) and translocation factor (TF) were determined. Meanwhile, to evaluate the level of pollution in the study area was used the contamination factor (CF), potential ecological risk factor (Eri) and the potential ecological risk index (RI). The order of heavy metals according to their concentration in the soil samples at both sampling sites was as follows: Fe > Ni > Zn > Pb. Concentrations in excess of the limits allowed for soils in the samples collected in the vicinity of the smelter were recorded for Pb (173.13 mg kg-1), Zn (1217.48 mg kg-1), and Ni (1443.93 mg kg-1), while at the control site, Zn (270.82 mg kg-1) and Ni (375.47 mg kg-1) were found in excess concentrations. But lead (Pb) level was under allowed limit. The data showed that the stinging nettle is not a hyperaccumulator because BCF < 1 at both sites for all metals under study. Furthermore, analysis of the translocation factor (TFsteam/root) showed that at low of heavy metal concentrations, their mobility was higher (TF > 1). The lowest mobility (TF < 1) was observed at site I (Poklek), where the concentration of heavy metals was higher, except for Fe. The opposite was shown for mobility of metals from stems to leaves (TFleave/steam). The evaluation of CF showed that the area near the Ferronikeli smelter had low degree of Pb, moderate degree of Zn and considerable degree of Ni contamination. The values of RI indicate low potential ecological risk index.

Towards defining the core Saccharum microbiome: input from five genotypes.

BACKGROUND: Plant microbiome and its manipulation inaugurate a new era for plant biotechnology with the potential to benefit sustainable crop production. Here, we used the large-scale 16S rDNA sequencing analysis to unravel the dynamic, structure, and composition of exophytic and endophytic microbial communities in two hybrid commercial cultivars of sugarcane (R570 and SP80-3280), two cultivated genotypes (Saccharum officinarum and Saccharum barberi) and one wild species (Saccharum spontaneum). RESULTS: Our analysis identified 1372 amplicon sequence variants (ASVs). The microbial communities' profiles are grouped by two, root and bulk soils and stem and leave when these four components are compared. However, PCoA-based data supports that endophytes and epiphytes communities form distinct groups, revealing an active host-derived mechanism to select the resident microbiota. A strong genotype-influence on the assembly of microbial communities in Saccharum ssp. is documented. A total of 220 ASVs persisted across plant cultivars and species. The ubiquitous bacteria are two potential beneficial bacteria, Acinetobacter ssp., and Serratia symbiotica. CONCLUSIONS: The results presented support the existence of common and cultivar-specific ASVs in two commercial hybrids, two cultivated canes and one species of Saccharum across tissues (leaves, stems, and roots). Also, evidence is provided that under the experimental conditions described here, each genotype bears its microbial community with little impact from the soil conditions, except in the root system. It remains to be demonstrated which aspect, genotype, environment or both, has the most significant impact on the microbial selection in sugarcane fields.

Environmental-biomechanical reciprocity and the evolution of plant material properties.

Abiotic-biotic interactions have shaped organic evolution since life first began. Abiotic factors influence growth, survival, and reproductive success, whereas biotic responses to abiotic factors have changed the physical environment (and indeed created new environments). This reciprocity is well illustrated by land plants who begin and end their existence in the same location while growing in size over the course of years or even millennia, during which environment factors change over many orders of magnitude. A biomechanical, ecological, and evolutionary perspective reveals that plants are (i) composed of materials (cells and tissues) that function as cellular solids (i.e. materials composed of one or more solid and fluid phases); (ii) that have evolved greater rigidity (as a consequence of chemical and structural changes in their solid phases); (iii) allowing for increases in body size and (iv) permitting acclimation to more physiologically and ecologically diverse and challenging habitats; which (v) have profoundly altered biotic as well as abiotic environmental factors (e.g. the creation of soils, carbon sequestration, and water cycles). A critical component of this evolutionary innovation is the extent to which mechanical perturbations have shaped plant form and function and how form and function have shaped ecological dynamics over the course of evolution.

Global Mercury Assimilation by Vegetation.

Assimilation of mercury (Hg) by vegetation represents one of the largest global environmental Hg mass fluxes. We estimate Hg assimilation by vegetation globally via a bottom-up scaling approach using tissue Hg concentrations synthesized from a comprehensive database multiplied by respective annual biomass production (NPP). As global annual NPP is close to annual vegetation die-off, Hg mass associated with global NPP approximates the transfer of Hg from plants to soils, which represents an estimate of vegetation-mediated atmospheric deposition. Annual vegetation assimilation of Hg from combined atmospheric and soil uptake is estimated at 3062 ± 607 Mg yr-1, which is composed of 2491 ± 551 Mg yr-1 from aboveground tissue uptake and 571 ± 253 Mg yr-1 from root uptake. Assimilation of atmospheric Hg amounts to 2422 ± 483 Mg yr-1 when considering aboveground tissues only. Atmospheric assimilation increases to 2705 ± 504 Mg yr-1 when considering that root Hg may be partially derived from prior foliar uptake and transported internally to roots. Estimated atmospheric Hg assimilation by vegetation is 54-137% larger than the current model and litterfall estimates, largely because of the inclusion of lichens, mosses, and woody tissues in deposition and all global biomes. Belowground, about 50% of root Hg was taken up from soils with currently unknown ecological and biogeochemical consequences.

Mass spectrometry imaging for direct visualization of components in plants tissues.

Mass spectrometry is considered the most informative technique for components identification and has been widely adopted in plant sciences. However, the spatial distribution of compounds in the plant, which is vital for the exploration of plant physiological mechanisms, is missed in MS analysis. In recent years, mass spectrometry imaging has brought a great breakthrough in plant analysis because it can determine both the molecular compositions and spatial distributions, which is conducive to understand functions and regulation pathways of specific components in plants. Mass spectrometry imaging analysis of plant tissue is toward high sensitivity, high spatial resolution, and even single-cell analysis. Despite many challenges and technical barriers, such as difficulties of sample pretreatment caused by morphological diversity of plant tissues, obstacles for high spatial resolution imaging, and so on, lots of researches have contributed to remarkable progress, including improvement in tissue preparation, matrix innovation, and ionization mode development. This review focuses on the advances of mass spectrometry imaging analysis of plants in the last 5 years, including commonly used ionization techniques, technical advances, and recent applications of mass spectrometry imaging in plants.

Organophosphate pesticides sequestered in tissues of a seagrass species - Zostera capensis from a polluted watershed.

Organophosphate pesticides (OPPs) are persistent in the environment, but little information is available on their bioaccumulation in seagrass. In this study, the seagrass - Zostera capensis was collected from Swartkops Estuary in South Africa to investigate the bioaccumulation of OPPs from contaminated sediments and the water column. This plant was chosen because it grows abundantly in the estuary's intertidal zone, making it a viable phytoremediator in the urban environment. Extraction was performed by the QuEChERS method followed by GC-MS analysis. The mean concentration of ∑OPPs ranged from 0.01 to 0.03 µg/L for surface water; 6.20-13.35 µg/kg dw for deep-rooted sediments; 18.79-37.75 µg/kg dw for leaf tissues and 12.14-39.80 µg/kg dw for root tissues of Z. capensis. The biota-sediment accumulation factors (BSAFs) were greater than one, indicating the potential for Z. capensis to bioaccumulate and intercept the targeted pesticides. A weak insignificant correlation observed between log BSAFs and log Kow indicates that the bioaccumulation of OPPs in tissues of Z. capensis were not dependent on the Kow. Eight of the selected pesticides had root-leaf translocation factors (TFr-l) greater than 1, indicating that Z. capensis can transport these chemicals from roots to leaves. The results from this study implies that this plant species can clean up OPP contamination in the environment.

Biomonitoring of heavy metal contamination with roadside trees from metropolitan area of Hefei, China.

Air and dust borne heavy metals can be deposited and bioaccumulated by plants; therefore, biomonitoring employing plants is an effective tool for environmental impact assessment in urban environments. In this study, in addition to road dust, leaves and bark were collected from four common tree species at roadside and urban park sampling sites within the metropolitan area of Hefei, China. A range of heavy metals were analyzed by ICP-MS and AFS. The metal accumulation index (MAI) was adopted to compare the bioaccumulation capacity. Results showed that Cd was highly enriched in road dust although its abundance was low in comparison with that of other elements. The MAI values presented a narrow range (1.8-2.7); however, significant differences (p < 0.05) were found for Al, Cu, Zn, and As among the tree species. Moreover, deciduous Platanus orientalis bioaccumulated more nonessential As than the other species and deserved further risk management. In addition, bark samples from Cinnamomum camphora bioaccumulated more heavy metals than the other species as a result of its morphological and anatomical characteristics. The distribution patterns of heavy metals in tree tissues showed obvious spatial heterogeneity, as impacted by anthropogenic activities to varying degrees. This study examined the biomonitoring potential of roadside trees and the distribution pattern of heavy metals in an urban area under rapid development. Results from the present study could provide baseline data for urban environmental impact assessment and the design of green belts.

Study of Potentially Toxic Elements Uptake into Organs of Quercus spp. from Copper Deposits in Slovakia, Italy and Portugal.

The article is focused on the application of Energy dispersive micro X-ray fluorescence spectroscopy as a specific method to determine the contents of potentially toxic elements and its spread in plant tissues. As a model species, Quercus spp. were selected. In order to compare the obtained results with previous research, four well-described abandoned Cu-deposits were selected for sampling: Lubietová (Slovakia), Libiola and Caporciano (Italy), and São Domingos (Portugal). The results of micro X-ray fluorescence spectrometry confirm the irregular contamination of Quercus spp. by potentially toxic elements. The level of contamination is the highest predominantly in the root cortex, where is also the highest Ca contents (with exception of São Domingos). At Lubietová and Caporciano, high Ni content was described in branches cortex, in branches mesoderm also Fe, Cu and Zn. At the same time, the inhibition influence of Ca was also confirmed regarding the input of these elements into plants.

Mathematical Modelling of Auxin Transport in Plant Tissues: Flux Meets Signalling and Growth.

Plant hormone auxin has critical roles in plant growth, dependent on its heterogeneous distribution in plant tissues. Exactly how auxin transport and developmental processes such as growth coordinate to achieve the precise patterns of auxin observed experimentally is not well understood. Here we use mathematical modelling to examine the interplay between auxin dynamics and growth and their contribution to formation of patterns in auxin distribution in plant tissues. Mathematical models describing the auxin-related signalling pathway, PIN and AUX1 dynamics, auxin transport, and cell growth in plant tissues are derived. A key assumption of our models is the regulation of PIN proteins by the auxin-responsive ARF-Aux/IAA signalling pathway, with upregulation of PIN biosynthesis by ARFs. Models are analysed and solved numerically to examine the long-time behaviour and auxin distribution. Changes in auxin-related signalling processes are shown to be able to trigger transition between passage- and spot-type patterns in auxin distribution. The model was also shown to be able to generate isolated cells with oscillatory dynamics in levels of components of the auxin signalling pathway which could explain oscillations in levels of ARF targets that have been observed experimentally. Cell growth was shown to have influence on PIN polarisation and determination of auxin distribution patterns. Numerical simulation results indicate that auxin-related signalling processes can explain the different patterns in auxin distributions observed in plant tissues, whereas the interplay between auxin transport and growth can explain the 'reverse-fountain' pattern in auxin distribution observed at plant root tips.

A rapid and universal method for isolating starch granules in plant tissues.

Starch is the major form of carbohydrate storage in plants and exists as discrete starch granules (SGs). Isolation of high-quality SGs in different plant tissues is a prerequisite for studying the roles of SGs during plant growth, development, and responses to abiotic stress. However, it is difficult to isolate transitory SGs from leaves and storage SGs from pollen grains due to their small sizes and low quantities. Herein, we develop a novel method for isolating SGs by using the aqueous two-phase system (ATS) of ethanol/NaH2 PO4 . The ATS method efficiently separated SGs from contaminants based on their differences in density, solubility, and polarity. Using this method, we first isolated and purified three kinds of SGs from maize seeds, pollen, and leaves. The biochemical, microscopic, and proteomic analyses demonstrated the high purity of the isolated SGs. Proteomic analysis revealed distinct differences in SG-bound proteins between seed SGs and pollen SGs. As a simple, rapid, and low-cost method, the ATS-based method exhibits highly universal and reproducible results for starch-containing tissues in various plant species.

Effect of Thermal and Nonthermal Processing on Textural Quality of Plant Tissues.

In the current fast revolving world, the consumption of processed food is increasing drastically. The population who depend on these processed foods are also cautious about the quality and safety of what they consume. This being the case, in order to satisfy the consumer it is the responsibility of the researcher and the manufacturer to check what happens to food on processing. Plant-derived foods such as fruits and vegetables are sensitive producers which are to be handled cautiously through each steps involved in processing, starting from harvest to storage, processing to package, transportation to distribution, till it reaches the consumer. During processing, the plant materials, which are made up of complex structural components such as lignin, cellulose, pectin, etc. undergo changes which has its effect on the quality attributes of the final product. Texture is an important quality parameter of all the sensory properties. The relation between the structure of the plant tissue and the texture of the final product is reviewed in this paper comprehensively.

A simple method for RNA isolation from various tissues of the tree Neolamarckia cadamba.

Plant tissues contain abundant polysaccharides, phenolic compounds and other metabolites, which makes it difficult to isolate high-quality RNA from them. In addition, Neolamarckia cadamba contains large quantities of other components, particularly RNA-binding alkaloids, which makes the isolation even more challenging. Here, we describe a concise and efficient RNA isolation method that combines the cetyltrimethyl ammonium bromide (CTAB) and Plant RNA Kit (Omega) protocols. Gel electrophoresis showed that RNA extracted from all tissues, using this protocol, was of good integrity and without DNA contamination. Furthermore, the isolated RNA was of high purity, with an A260/A280 ratio of 2.1 and an A260/A230 ratio of >2.0. The isolated RNA was also suitable for downstream applications, such as reverse transcription-polymerase chain reaction (RT-PCR) and quantitative RT-PCR (RT-qPCR). The RNA isolation method was also efficient for recalcitrant plant tissues.

Molecular detection of bacteria in plant tissues, using universal 16S ribosomal DNA degenerated primers.

Highly specific, sensitive and rapid tests are required for the detection and identification of covert bacterial contaminations in plant tissue cultures. Current methods available for this purpose are tedious, time consuming, highly error prone, expensive, require advanced technical expertise and are sometimes ineffective. We report here the development of a sensitive polymerase chain reaction (PCR) based method for the rapid detection and identification of bacteria occurring in plant tissue cultures. A total of 121 16S ribosomal DNA (rDNA) coding regions from 14 different groups of bacteria, algae and plants, available in the Gene Bank/European Molecular Biology Laboratory databases, were aligned and several conserved DNA sequences of bacterial origin were identified. From those, five degenerated primers were designed in order to amplify only the bacterial DNA present in mixed plant/bacteria genomic DNA extracts. A known amount of bacterial suspension of either covert Pseudomonas or covert Bacillus were added to in vitro plant leaves and total plant/bacterial DNA extracted using three different methods to determine the lowest number of bacteria required to be present in order to allow their detection. The highest sensitivity of the bacterial cell detection was 2.5 × 106 cells of both Bacillus and Pseudomonas inoculums, using template DNA prepared by the MiniPrep method. Generation of PCR amplification fragments was achieved only for the 16S rDNA bacterial gene by using four combinations of degenerated primers. Successive sequence analysis of these amplified fragments led to the rapid detection and molecular identification of bacteria covertly associated with plants.

Optimized extraction methodology for phenolic compounds in soil and plant tissues: Their implications in plant growth and gall formation.

Phenolic compounds, abundant secondary metabolites in plants, profoundly influence soil ecosystems, plant growth, and interactions with herbivores. Phenolic in soil microorganisms have the potential to impact a wide range of activities in plant-soil interactions. However, the existing methods for measuring microbial activity are typically time-consuming, intricate, and expensive. In this study, we propose modifications to the method used for the extraction and quantification of various types of phenolics in soil and plant tissues. There have been substantial advancements in research aimed at extracting, identifying, and quantifying phenolic compounds in the plant and soil samples. This study discusses the use of different methodologies in the analysis of phenolic compounds. In addition, we investigated the effect of phenolics on plant growth and cues in gall-forming under environmental disturbances.•This method is the optimum way to extract phenolic from soil and microbial activity in bulk and rhizosphere soil.•It can be used on any soil type and plant tissue, metabolites extracted from living organisms.

Untargeted lipidomic profiling of grapes highlights the importance of modified lipid species beyond the traditional compound classes.

The aim of this paper is to provide a detailed characterisation of grape lipidome. To achieve this objective, it starts by describing a pipeline implemented in R software to allow the semi-automatic annotation of the detected lipid species. It also provides an extensive description of the different properties of each molecule (such as retention time dependencies, mass accuracy, adduct formation and fragmentation patterns), which allowed the annotations to be made more accurately. Most annotated lipids in the grape samples were (lyso)glycerophospholipids and glycerolipids, although a few free fatty acids, hydroxyceramides and sitosterol esters were also observed. The proposed pipeline also allowed the identification of a series of methylated glycerophosphates never previously observed in grapes. The current results highlight the importance of expanding chemical analyses beyond the classical lipid categories.

A typical method for decellularization of plants as biomaterials.

Decellularization is a process by which cells are removed from tissues or organs, leaving behind the extracellular matrix (ECM) structure. This process has gained interest in the fields of tissue engineering and regenerative medicine as a way to prepare suitable scaffolds for tissue reconstruction. Although the initial efforts come with the animal tissues, this technique can also be applied to various plant tissues with simple modifications, as plant-derived biomaterials have the benefit of being biocompatible and serving as a safe, all-natural substitute for synthetic or animal originated materials. Additionally, plant-derived biomaterials may help cells grow and differentiate, creating a three-dimensional environment for tissue regeneration and repair. Here we demonstrate a general method for plant tissue decellularization, including already experienced approaches and techniques.•Exhibit the basic steps for plant decellularization, which may be applied to several other plant tissues.•The proposed approach may be optimized considering various intended uses.•Gives basic information for the determination of decellularization efficiency.

Maize Internode Autofluorescence at the Macroscopic Scale: Image Representation and Principal Component Analysis of a Series of Large Multispectral Images.

A quantitative histology of maize stems is needed to study the role of tissue and of their chemical composition in plant development and in their end-use quality. In the present work, a new methodology is proposed to show and quantify the spatial variability of tissue composition in plant organs and to statistically compare different samples accounting for biological variability. Multispectral UV/visible autofluorescence imaging was used to acquire a macroscale image series based on the fluorescence of phenolic compounds in the cell wall. A series of 40 multispectral large images of a whole internode section taken from four maize inbred lines were compared. The series consisted of more than 1 billion pixels and 11 autofluorescence channels. Principal Component Analysis was adapted and named large PCA and score image montages at different scales were built. Large PCA score distributions were proposed as quantitative features to compare the inbred lines. Variations in the tissue fluorescence were clearly displayed in the score images. General intensity variations were identified. Rind vascular bundles were differentiated from other tissues due to their lignin fluorescence after visible excitation, while variations within the pith parenchyma were shown via UV fluorescence. They depended on the inbred line, as revealed by the first four large PCA score distributions. Autofluorescence macroscopy combined with an adapted analysis of a series of large images is promising for the investigation of the spatial heterogeneity of tissue composition between and within organ sections. The method is easy to implement and can be easily extended to other multi-hyperspectral imaging techniques. The score distributions enable a global comparison of the images and an analysis of the inbred lines' effect. The interpretation of the tissue autofluorescence needs to be further investigated by using complementary spatially resolved techniques.

The Use of Flow Cytometry for Estimating Genome Sizes and DNA Ploidy Levels in Plants.

Flow cytometry has emerged as a uniquely flexible, accurate, and widely applicable technology for the analysis of plant cells. One of its most important applications centers on the measurement of nuclear DNA contents. This chapter describes the essential features of this measurement, outlining the overall methods and strategies, but going on to provide a wealth of technical details to ensure the most accurate and reproducible results. The chapter is aimed to be equally accessible to experienced plant cytometrists as well as those newly entering the field. Besides providing a step-by-step guide for estimating genome sizes and DNA-ploidy levels from fresh tissues, special attention is paid to the use of seeds and desiccated tissues for such purposes. Methodological aspects regarding field sampling, transport, and storage of plant material are also given in detail. Finally, troubleshooting information for the most common problems that may arise during the application of these methods is provided.