Intact Plants Research Articles

Hyperspectral imaging for pest symptom detection in bell pepper

BackgroundThe automation of pest monitoring is highly important for enhancing integrated pest management in practice. In this context, advanced technologies are becoming increasingly explored. Hyperspectral imaging (HSI) is a technique that has been used frequently in recent years in the context of natural science, and the successful detection of several fungal diseases and some pests has been reported. Various automated measures and image analysis methods offer great potential for enhancing monitoring in practice.ResultsIn this study, the use of hyperspectral imaging over a wide spectrum from 400 to 2500 nm is investigated for noninvasive identification and the distinction of healthy plants and plants infested with Myzus persicae (Sulzer) and Frankliniella occidentalis (Pergande) on bell peppers. Pest infestations were carried out in netted areas, and images of single plants and dissected leaves were used to train the decision algorithm. Additionally, a specially modified spraying robot was converted into an autonomous platform used to carry the hyperspectral imaging system to take images under greenhouse conditions. The algorithm was developed via the XGBoost framework with gradient-boosted trees. Signals from specific wavelengths were found to be associated with the damage patterns of different insects. Under confined conditions, M. persicae and F. occidentalis infestations were distinguished from each other and from the uninfested control for single leaves. Differentiation was still possible when small whole plants were used. However, application under greenhouse conditions did not result in a good fit compared to the results of manual monitoring.ConclusionHyperspectral images can be used to distinguish sucking pests on bell peppers on the basis of single leaves and intact potted bell pepper plants under controlled conditions. Wavelength reduction methods offer options for multispectral camera usage in high-grown vegetable greenhouses. The application of automated platforms similar to the one tested in this study could be possible, but for successful pest detection under greenhouse conditions, algorithms should be further developed fully considering real-world conditions.

Warming triggers stomatal opening by enhancement ofphotosynthesis and ensuing guard cell CO2 sensing,whereashigher temperatures induce a photosynthesis-uncoupled response.

Plants integrate environmental stimuli to optimize photosynthesis vs water loss by controlling stomatal apertures. However, stomatal responses to temperature elevation and the underlying molecular genetic mechanisms remain less studied. We developed an approach for clamping leaf-to-air vapor pressure difference (VPDleaf) to fixed values, and recorded robust reversible warming-induced stomatal opening in intact plants. We analyzed stomatal temperature responses of mutants impaired in guard cell signaling pathways for blue light, abscisic acid (ABA), CO2, and the temperature-sensitive proteins, Phytochrome B (phyB) and EARLY-FLOWERING-3 (ELF3). We confirmed that phot1-5/phot2-1 leaves lacking blue-light photoreceptors showed partially reduced warming-induced stomatal opening. Furthermore, ABA-biosynthesis, phyB, and ELF3 were not essential for the stomatal warming response. Strikingly, Arabidopsis (dicot) and Brachypodium distachyon (monocot) mutants lacking guard cell CO2 sensors and signaling mechanisms, including ht1, mpk12/mpk4-gc, and cbc1/cbc2 abolished the stomatal warming response, suggesting a conserved mechanism across diverse plant lineages. Moreover, warming rapidly stimulated photosynthesis, resulting in a reduction in intercellular (CO2). Interestingly, further enhancing heat stress caused stomatal opening uncoupled from photosynthesis. We provide genetic and physiological evidence that the stomatal warming response is triggered by increased CO2 assimilation and stomatal CO2 sensing. Additionally, increasing heat stress functions via a distinct photosynthesis-uncoupled stomatal openingpathway.

Structure-Dependent uptake and metabolism of Tire additives Benzothiazoles in carrot plant

Tire additives, such as benzothiazole and its derivatives (collectively called BTs), are large-volume chemicals that are constantly emitted into agricultural environment via tire-road wearing and other actions. The potential accumulation of BTs in food crops depends largely on their metabolism in plants, which is poorly understood. Herein, we evaluated uptake and metabolism of six BTs in carrot callus and intact carrot plants to understand their structure-specific metabolism. All BTs were readily taken up by carrot roots, with their root concentration factors (RCF) ranging from 1.66 ± 0.01 to 2.95 ± 0.05. Although the tested BTs exhibited poor upward translocation from root to leaves (translocation factors < 1), the translocation factors of 2-methylbenzothiazole (0.79) and 2-aminobenzothiazole (0.65) were significantly higher than that of 2-methylbenzothiazole (0.18) and 2-(methylthio)benzothiazole (0.22). These results indicated the structure-dependent uptake and translocation of BTs in carrot. Correlation analysis between log Kow and log RCF or TF revealed that the hydrophobicity of BTs predominantly affected their root uptake and acropetal translocation in carrots. With the aid of high-resolution mass spectrometry, a total of 18 novel metabolites of BTs were tentatively identified, suggesting that BT compounds can be metabolized by carrot callus. The proposed metabolites of BTs include four hydroxylated products, one demethylated product, five glycosylated products and eight amino acid conjugated products, revealing that glycosylation and amino acid conjugation were the dominant transformation pathways for BT metabolism in carrot. However, the detected species of metabolites for six BTs varied distinctly, indicating structure-specific metabolism of BTs in plants. The findings of this study improve our understanding of structure-dependent fate and transformation of BTs in plants. Since BTs metabolites in food crops could present an unintended exposure route to consumers, the structure-specific differences of BTs uptake, metabolism and accumulation in plants must be considered when addressing human dietary exposure risks.

A 3D-Printable smartphone accessory for plant leaf chlorophyll measurement

Plant health and nutrition are universally inferred from leaf chlorophyll content. This research developed a 3D-printed accessory which attaches to the ambient light sensor in a smartphone to estimate leaf chlorophyll content in five tropical plant species. It unveils 3D printing files and assembling details to freely built the accessory anywhere. It is made from a 3D-printed body, a lighting circuit and common spare parts to measure a 663 nm LED band transmission through intact plant leaves. This chlorophyll absorbing light band allows to measure its concentration. The device was tested by comparing its readings to the universal spectrophotometric test or by leaf parallel measurements with a standard SPAD 502™ meter, and it performed as well as these universal standard methods. Due to well-studied relationships between chlorophyll concentration and nutritional status of plants, and the ubiquitous presence of other sensors in smartphones today, the independent improvement and adoption of this smartphone-connected system would ease the spread of precision farming and digital agronomy practices throughout the different scales of agriculture.

Emissions of volatile organic compounds from aboveground and belowground parts of rapeseed (Brassica napus L.) and tomato (Solanum lycopersicum L.)

Root systems represent a source of Volatile Organic Compounds (VOCs) that may significantly contribute to the atmospheric VOC emissions from agroecosystems and shape soil microbial activity. To gain deeper insights into the role of roots in the VOC emissions from crops, we developed a dynamic chamber with isolated aboveground and belowground compartments, allowing for simultaneous measurements of VOC fluxes from both compartments in controlled conditions. We continuously monitored VOC emissions from intact plants of rapeseed (Brassica napus L.) and tomato (Solanum lycopersicum L.) i) over 24 h when plants were rooted in soil, and ii) over 6 h following soil removal. The measurements were performed using a highly sensitive Proton Transfer Reaction – Time of Flight – Mass Spectrometer and a Thermic Desorption- Gas Chromatography – Mass Spectrometer. Net VOC emissions measured at the soil surface represented <5 % of the aboveground emissions and were higher during the day than at night. However, when soil was removed, belowground VOC emissions became up to two times higher than aboveground emissions. This large increase in VOC emissions from roots observed after soil removal was almost exclusively due to methanol emissions. Differences in VOC composition between plant species were also detected with and without soil: rapeseed emitted more sulphurous and nitrogenous compounds and tomato more mono- and poly-unsaturated hydrocarbons. Our results suggest that roots may be a largely underestimated VOC source and that the soil is a strong sink for root-borne methanol. Root VOC emissions should be considered when agricultural practices involve roots excavation.

Functional characterization of a novel terpene synthase GaTPS1 involved in (E)-α-bergamotene biosynthesis in Gossypium arboreum

Terpenoids in plants are mainly synthesized by terpene synthases (TPSs), which play an important role in plant-environment interactions. Gossypium arboreum is one of the important cotton cultivars with excellent pest resistance, however, the biosynthesis of most terpenoids in this plant remains unknown. In this study, we performed a comparative transcriptome analysis of leaves from intact and Helicoverpa armigera-infested cotton plants. The results showed that the H. armigera infestation mainly induced the JA signaling pathway, ten TPS genes were differentially expressed in G. arboreum leaves. Among them, a novel terpene synthase, GaTPS1, was heterologously expressed and functionally characterized in vitro. The enzymatic reaction indicated that recombinant GaTPS1 was primarily responsible for the production of (E)-α-bergamotene. Moreover, molecular docking and site-directed mutagenesis analysis demonstrated that two amino acid residues, A412L and Y535F, distinctly influenced the catalytic activities and product specificity of GaTPS1. The mutants GaTPS1-A412L and GaTPS1-Y535F resulted in a decrease in the proportion of products (E)-α-bergamotene and D-limonene, while an increase in the proportion of products (E)-β-farnesene, α-pinene and β-myrcene. Our findings provide valuable insights into understanding the molecular basis of terpenoid diversity in G. arboreum, with potential applications in plant metabolism regulation and the improvement of resistant cotton cultivars.

Seasonal variability and seagrass traits affect methane fluxes in a subtropical meadow

Abstract Plant traits which vary both within and between species often drive biogeochemical cycling. Understanding the relative role of within‐ and between‐species trait variability in driving carbon cycling is essential to scaling site measurements to global carbon budgets. In seagrass meadows, carbon and nitrogen mineralization rates and associated greenhouse gas emissions are highly variable, impeding our ability to reliably predict whether meadows are net carbon sinks. Evaluating the influence of within‐ and between‐species trait variability on greenhouse gas fluxes will improve our understanding of local‐scale drivers of greenhouse gas production and consumption in seagrass meadows. To test the effects of plant traits on dissolved greenhouse gas fluxes, we performed mesocosm incubations with live, intact seagrass plants. We compared methane (CH4) and nitrous oxide (N2O) fluxes under dark and light conditions from sediments dominated by Halodule wrightii and Thalassia testudinum across dormant, early and peak growing seasons in a subtropical meadow along the west coast of peninsular Florida. We also measured oxygen (O2) fluxes to interpret greenhouse gas fluxes within the context of community metabolism. We measured several seagrass traits, such as above‐ and below‐ground biomass and leaf and root area and assessed their impact as well as the impact of species identity on dissolved gas fluxes. We found that abiotic factors linked to metabolism (i.e. light and temperature) influenced greenhouse gas fluxes across seasons. In addition to light conditions and sampling month, plant size (a composite trait variable) was a significant predictor of O2 consumption and CH4 production under dark conditions, and better predicted fluxes than individual plant traits. CH4 production was slightly higher in H. wrightii‐dominated sediments, but species identity was less important than plant size in driving CH4 production. N2O fluxes were low and not influenced by plant traits or species identity. Synthesis: Our results indicate that within‐species more so than between‐species trait variability drives the direction and magnitude of CH4 fluxes in seagrass meadows. We identified a trade‐off where seagrass biomass is often associated with enhanced sediment carbon storage, but in our study, plant size promoted CH4 production, potentially offsetting the benefits of long‐term storage.

Resprouting Control of Ailanthus altissima by Means of Cut and Stump Covering: Experimental Evidence for a Promising Technique

Ailanthus altissima is an invasive tree threatening the biodiversity in natural and disturbed habitats of temperate regions, primarily because of its high resprouting vigor. To test the effect of light exclusion on stem and root sprouting, black covers were applied on the cut stumps of 3-year-old saplings grown in pots. The treatments were as follows: SL, stumps exposed to light; SH, only stumps covered; SPH, the entire pot covered; C, intact control plants. The covers were removed when the root sprouts appeared. The emergence of stem and root sprouts was checked weekly, and their number and biomass were determined at the growth peak (cut I and III) and the end (cut II and IV) of two seasons after cutting. Stem sprouts were produced only in the first growth season in SL and SH and never in SPH and the controls. The number and biomass of the root sprouts were the highest in SH at cut I and in SL in the following cuts. In SH, the sprouting ceased completely after cut I, suggesting a rapid consumption of root reserves, while in SL and SPH, repeated cutting progressively reduced the sprouting vigor. We concluded that covering the stems after cutting is a promising technique to control ailanthus resprouting without the use of herbicides.

Improving the Salt Tolerance of “Old Limachino Tomato” by Using a New Salt-Tolerant Rootstock

Salinity is a major constraint limiting the yield of tomatoes. However, grafting strategies may help to overcome the salt toxicity of this important horticultural species if appropriate rootstocks are identified. The present study aimed to test a new rootstock, JUPAFORT1, obtained by crossing the glycophyte Solanum lycopersicum (cv. Poncho Negro) with the halophyte wild-related species Solanum chilense to improve the salinity tolerance of the Chilean tomato landrace Old Limachino Tomato (OLT). Intact OLT plants were exposed to 0, 80, or 160 mM of NaCl for 21 days at the vegetative stage and compared with self-grafted (L/L) and Limachino plants grafted on JUPAFORT1 rootstock (L/R) under a completely randomized design. JUPAFORT1 increased OLT scion vigor in the absence of salt but did not significantly increase fresh weight under stress conditions. However, JUPAFORT1 confers to the scion an anisohydric behavior contrasting with the isohydric behavior of L and L/L plants as indicated by measurements of stomatal conductance; L/R plants were able to maintain their metabolic status despite a slight decrease in the leaf’s relative water content. JUPAFORT1 rootstock also enabled the maintenance of photosynthetic pigment concentrations in the scion in contrast to L and L/L plants, which exhibited a decrease in photosynthetic pigments under stress conditions. L/R plants encountered oxidative stress at the highest stress intensity (160 mM of NaCl) only, while L and L/L plants suffered from oxidative damage at a lower dose (80 mM of NaCl). L/R plants behaved as includer plants and did not sequester Na+ in the root system, in contrast to L and L/L, which behaved as excluder plants retaining Na+ in the root system to avoid its translocation to the shoots. The expression of genes coding for ion transporters (HKT1.1, HKT1.2, LKT1, SKOR, SOS2, and SOS3) in the root system was not modified by salinity in L/R. In contrast, their expression varied in response to salinity in L and L/L. Overall, L/R plants exhibited higher physiological stability than L/L or L plants in response to an increasing NaCl dose and did not require additional energy investment to trigger an adaptative response to salinity. This suggests that the constitutive salinity tolerance of the halophyte S. chilense was maintained in the interspecific rootstock. JUPAFORT1 issued from S. lycopersicum x S. chilense may thus improve salt-stress resilience in OLT tomatoes. Additional studies are required to identify the molecular components involved in the root-to-shoot signaling pathway in this promising material.

Absorption and Xylem Transport of 33P-Labeled Phosphorus in Nodulated Soybean Plants

Phosphorus (P) is an essential macro-element for plants, and understanding the characteristics of the absorption and transport of P in crops is essential. The low availability of P restricts the growth, nitrogen fixation, and yield of soybean plants. In this research, the radioisotope 33P was supplied to the culture solution to trace the absorption and transport of P in nodulated soybean plants monitored using an imaging plate. The absorption rate of 33P was almost the same under the light and dark conditions. The absorption rate of 33P in the decapitated roots was near to that of the intact plants under light. These results indicate that the P absorption is not affected by evapotranspiration over a short time period. Conversely, the 33P transport from the roots to the shoot was significantly lower under dark conditions than it was under light conditions, although some 33P reached the top of the shoots under both the light and dark conditions. The transport of P to the shoots depends on the transpiration supplemented by the root pressure. The multiplication value of the 33P concentration in the xylem sap and transpiration rate was almost equivalent to the transport rate of 33P in the intact shoots. This value may be adaptable and used to estimate the transport rate of P for the diagnosis.

The extremotolerant desert moss Syntrichia caninervis is a promising pioneer plant for colonizing extraterrestrial environments

Many plans to establish human settlements on other planets focus on adapting crops to growth in controlled environments. However, these settlements will also require pioneer plants that can grow in the soils and harsh conditions found in extraterrestrial environments, such as those on Mars. Here, we report the extraordinary environmental resilience of Syntrichia caninervis, a desert moss that thrives in various extreme environments. S.caninervis has remarkable desiccation tolerance; even after losing >98% of its cellular water content, it can recover photosynthetic and physiological activities within seconds after rehydration. Intact plants can tolerate ultra-low temperatures and regenerate even after being stored in a freezer at -80°C for 5 years or in liquid nitrogen for 1month. S.caninervis also has super-resistance to gamma irradiation and can survive and maintain vitality in simulated Mars conditions; i.e., when simultaneously exposed to an anoxic atmosphere, extreme desiccation, low temperatures, and intense UV radiation. Our study shows that S.caninervis is among the most stress tolerant organisms. This work provides fundamental insights into the multi-stress tolerance of the desert moss S.caninervis, a promising candidate pioneer plant for colonizing extraterrestrial environments, laying the foundation for building biologically sustainable human habitats beyond Earth.

Live Imaging of the Shoot Apical Meristem of Intact, Soil-Grown, Flowering Arabidopsis Plants.

All aerial organs in plants originate from the shoot apical meristem, a specialized tissue at the tip of a plant, enclosing a few stem cells. Understanding developmental dynamics within this tissue in relation to internal and external stimuli is of crucial importance. Imaging the meristem at the cellular level beyond very early stages requires the apex to be detached from the plant body, a procedure that does not allow studies in living, intact plants over longer periods. This protocol describes a new confocal microscopy method with the potential to image the shoot apical meristem of an intact, soil-grown, flowering Arabidopsis plant over several days. The setup opens new avenues to study apical stem cells, their interconnection with the whole plant, and their responses to environmental stimuli. Key features • Novel dissection and imaging method of the shoot apical meristem of Arabidopsis. • Procedure performed with intact, soil-grown, flowering plants. • Possibility of long-term live imaging of the shoot apical meristem. • Protocol can be adapted to different plant species.

DNA Delivery by Virus-Like Nanocarriers in Plant Cells.

Tobacco mild green mosaic virus (TMGMV)-like nanocarriers were designed for gene delivery to plant cells. High aspect ratio TMGMVs were coated with a polycationic biopolymer, poly(allylamine) hydrochloride (PAH), to generate highly charged nanomaterials (TMGMV-PAH; 56.20 ± 4.7 mV) that efficiently load (1:6 TMGMV:DNA mass ratio) and deliver single-stranded and plasmid DNA to plant cells. The TMGMV-PAH were taken up through energy-independent mechanisms in Arabidopsis protoplasts. TMGMV-PAH delivered a plasmid DNA encoding a green fluorescent protein (GFP) to the protoplast nucleus (70% viability), as evidenced by GFP expression using confocal microscopy and Western blot analysis. TMGMV-PAH were inactivated (iTMGMV-PAH) using UV cross-linking to prevent systemic infection in intact plants. Inactivated iTMGMV-PAH-mediated pDNA delivery and gene expression of GFP in vivo was determined using confocal microscopy and RT-qPCR. Virus-like nanocarrier-mediated gene delivery can act as a facile and biocompatible tool for advancing genetic engineering in plants.

Carbon nanotubes functionalized with α-aminoisobutyric acid-containing peptide increase gene delivery efficiency in plant mitochondria

Functionalized carbon nanotubes have shown tremendous promise in the field of plant biotechnology for genetic engineering and cargo delivery; recent findings have shown that they can be delivered within specific organelles, such as mitochondria and chloroplasts, in intact plants. 2-Aminoisobutyric acid is an unnatural amino acid that promotes helical conformation and has been demonstrated to increase membrane permeability. Rational substitution of this amino acid into a mitochondrial targeting peptide induced a helical conformation that, when functionalized onto polymer-coated carbon nanotubes, conferred increased membrane permeability compared with that of the native peptide. The secondary structure was maintained on the surface and, when used to deliver pDNA, led to an increase in gene expression, suggesting that this method may be used to enhance the delivery efficiency of existing functional peptides.

First Report of Leaf Spot Disease on Lonicera japonica Caused by Nigrospora oryzae in China.

Lonicera japonica Thunb. is a traditional Chinese medicinal plant, which widely cultivated in China, Japan and Korea. From August to October in 2021 and 2022, severe leaf spots symptoms were observed on L. japonica in medicinal botanical garden of Shandong University of Traditional Chinese Medicine (36°55'89"N, 116°79'91"E), Jinan, Shandong Province, China. The disease incidence was above 80% in the 25 acre cultivation area. Early symptoms were small brown spots on the leaves. Then the number of small spots gradually increased and spread over the entire leaves. The small brown spots seldom merge together to form larger lesions. Leaves with typical symptoms were collected from twenty individual plants, and cut into small 5×5 mm fragments in the junction of infected and healthy tissues. The fragments were sterilized in 75% ethanol for 30 s and 1% NaClO for 60 s, rinsed three times in sterile water, and then placed on potato dextrose agar (PDA). After 3 days of incubation at 25°C, fungal plugs along the edge of the colony were cut and transferred to new PDA for purification. A total number of 23 colonies with similar morphological characteristics were obtained, and three representative strains (Lj14, Lj18 and Lj20) were selected for subsequent study. The colonies grew rapidly on PDA and covered the entire petri dish in 4 days. Colonies had abundant aerial hyphae, initially white, round, later turning gray and black. Conidia were oblate or nearly spherical, single-celled, black, and measured in size from 9.6 to 13.2 μm × 7.9 to 16.1 μm in diameter (n=150) (Figure S1). The observed characteristics were close to those of Nigrospora spp. ( Wang et al. 2017). The genomic DNA was extracted, and PCR amplification of the rDNA internal transcribed spacer (ITS), β-tubulin gene (TUB), and translation elongation factor 1-alpha gene (TEF1) were completed by primers ITS1/ITS4, Bt2a/Bt2b and EF1-728F/EF1-986R (Carbone and Kohn, 1999). Sequences were deposited in GenBank (accession nos. OR936661, OR936662, OR936671 for ITS, OR947626, OR947627, OR947628 for TUB, and OR947629, OR947630, OR947631 for TEF1 sequences, respectively). BLAST analyses of ITS (OR936661), TUB(OR947626) and TEF1 (OR947629) sequences exhibited 100% (487 bp out of 487 bp), 99.48% (380 bp out of 382 bp), and 99.6% (248 bp out of 249 bp) similarity to the sequences of N. oryzae strains KoLRI_053384 (MZ855426), LC2991 (KY019496) and LC7307 (KY019409), respectively. Lj14, Lj18 and Lj20 formed a clade with N. oryzae LC6763 and LC2991 in phylogenetic tree (Figure S2). Based on morphological and molecular evidence, the pathogen was identified as N. oryzae (Berk. &Broome) Petch. To fulfill Koch's postulates, the pathogenicity was tested in vivo experiments. Thirty non-wounded healthy leaves of ten intact plants were inoculated with 10 µl spore suspension (106 spores/ml) of three strains, respectively. As negative control, thirty leaves of ten healthy plants were inoculated with sterile water. The inoculated plants were placed at 28°C in the growth chamber with high relative humidity. The pathogenicity tests were repeated three times. Distinct symptoms similar to that of natural conditions were observed on the leaves of inoculated plants after 4 to 7 days. The strain was reisolated from the lesions and identified as N. oryzae by morphological features and ITS sequence. The pathogen has been reported to cause leaf spot disease on tobacco (Wang et al. 2022) and asiatic dayflower (Qiu et al. 2022). To our knowledge, this is the first report of leaf spot caused by N. oryzae on Lonicera japonica in China. The research will be helpful for leaf spot disease control.

Plasmonics nanorod biosensor for in situ intracellular detection of gene expression biomarkers in intact plant systems

The intracellular developmental processes in plants, particularly concerning lignin polymer formation and biomass production are regulated by microRNAs (miRNAs). MiRNAs including miR397b are important for developing efficient and cost-effective biofuels. However, traditional methods of monitoring miRNA expression, like PCR, are time-consuming, require sample extraction, and lack spatial and temporal resolution, especially in real-world conditions. We present a novel approach using plasmonics nanosensing to monitor miRNA activity within living plant cells without sample extraction. Plasmonic biosensors using surface-enhanced Raman scattering (SERS) detection offer high sensitivity and precise molecular information. We used the Inverse Molecular Sentinel (iMS) biosensor on unique silver-coated gold nanorods (AuNR@Ag) with a high-aspect ratio to penetrate plant cell walls for detecting miR397b within intact living plant cells. MiR397b overexpression has shown promise in reducing lignin content. Thus, monitoring miR397b is essential for cost-effective biofuel generation. This study demonstrates the infiltration of nanorod iMS biosensors and detection of non-native miRNA 397b within plant cells for the first time. The investigation successfully demonstrates the localization of nanorod iMS biosensors through TEM and XRF-based elemental mapping for miRNA detection within plant cells of Nicotiana benthamiana. The study integrates shifted-excitation Raman difference spectroscopy (SERDS) to decrease background interference and enhance target signal extraction. In vivo SERDS testing confirms the dynamic detection of miR397b in Arabidopsis thaliana leaves after infiltration with iMS nanorods and miR397b target. This proof-of-concept study is an important stepping stone towards spatially resolved, intracellular miRNA mapping to monitor biomarkers and biological pathways for developing efficient renewable biofuel sources.

Microinjection of the CRISPR/Cas9 editing system through the germ pore of a wheat microspore induces mutations in the target Ms2 gene.

Microinjection is a direct procedure for delivering various compounds via micropipette into individual cells. Combined with the CRISPR/Cas9 editing technology, it has been used to produce genetically engineered animal cells. However, genetic micromanipulation of intact plant cells has been a relatively unexplored area of research,partly due to the cytological characteristics of these cells. This study aimed to gain insight into the genetic micromanipulation of wheat microspores using microinjection procedures combined with the CRISPR/Cas9 editing system targeting the Ms2 gene. Microspores were first reprogrammed by starvation and heat shock treatment to make them structurally suitable for microinjection. The large central vacuole was fragmented and the nucleus with cytoplasm was positioned in the center of the cell. This step and an additional maltose gradient provided an adequate source of intact single cells in the three wheat genotypes. The microcapillary was inserted into the cell through the germ pore to deliver a working solution with a fluorescent marker. This procedure was much more efficient and less harmful to the microspore than inserting the microcapillary through the cell wall. The CRISPR/Cas9 binary vectors injected into reprogrammed microspores induced mutations in the target Ms2 gene with deletions ranging from 1 to 16bp. This is the first report of successful genome editing in an intact microspore/wheat cell using the microinjection technique and the CRISPR/Cas9 editing system. The study presented offers a range of molecular and cellular biology tools that can aid in genetic micromanipulation and single-cell analysis.

ПРИМЕНЕНИЕ МИКРОСПЕКТРОФЛУОРИМЕТРА / МИКРОСПЕКТРОФОТОМЕТРА ДЛЯ ИССЛЕДОВАНИЯ ПОВЕРХНОСТИ РАСТИТЕЛЬНЫХ КЛЕТОК

The fluorescence and absorbance spectra of intact plant leaf cells from juniper Juniperus excelsa, Jerusalem-sage Phlomis tuberosa, butterbur Petasites spurius, centaury Centaurea scabiosa and wild chervil Anthriscus sylvestris have been obtained using the microspectrofluorimeter/ microspectrophotometer MSF-15 (LOMO, St. Petersburg). The obtained spectra of autofluorescence and absorbance made it possible to test the composition of compounds outside the cell wall. This tool is able to detect the presence of secretions on the surface of intact cells, including secondary metabolites with the fluorescence maxima in the region of 530-550 nm, characteristic of phenolic compounds. In the absorbance spectra of leaves, maxima were found in the region of 580-620 nm, which is peculiar to blue pigments azulenes. The presence of azulenes on the surface of cells has been confirmed by short-term extraction of these pigments with ethanol or acetone. Similar methodical approach has been recommended for rapid analysis of intact surfaces of plant cells.

Aquatic weed Eichhornia crassipes as sustainable feedstocks for biochar production: A potential of ammonium adsorption and kinetic models

The rapid emergence of unique types of pollutants is of worldwide concern owing to their deleterious effects on living beings. Mitigating pollution in the way of sustainability is the need of the hour to protect the existing environment from degradation. In this regard, eco-friendly biochar was prepared from the intact plant of Eichhornia crassipes (water hyacinth) without neglecting the roots, stems, leaves, and flowers and assessed for its ammonium removal efficiency in the study. Twelve biochars of two categories, viz., treated and normal, were produced under two different pyrolysis temperatures of 300 °C and 500 °C and three residence times of 30, 45, and 60 min. The adsorption experiments were conducted as batch studies in triplicate to examine the impacts of multiple adsorption-inducing factors in biochar. The maximum ammonium removal efficiency of about 83% was shown by the alkali-activated biochar produced at a higher temperature of 500 °C with a longer residence time of 60 min. The biochar showing higher ammonium adsorption was characterized by Scanning Electron Microscopy and Fourier Transform Infra-Red spectroscopy, which revealed the increased number of pores and the presence of C–O, OH, and CO groups that altogether aided the adsorption process. The kinetic study conducted with contact times of 30, 60, and 90 min showed that the linear regression values were >0.999 for the pseudo-second-order kinetic model, which revealed that the ammonia sorption of Eichhornia biochars followed a second-order mechanism of chemisorption. Synthesized biochar has shown significant efficacy in ammonia adsorption, making it worthwhile in applications where ammonia removal is necessary, particularly in wastewater treatment besides agriculture and air purification.

A protoplast-based transient gene expression assay for the identification of heat and oxidative stress-regulatory genes in perennial ryegrass

BackgroundWith the accumulating omics data, an efficient and time-saving transient assay to express target genes is desired. Mesophyll protoplasts, maintaining most stress-physiological responses and cellular activities as intact plants, offer an alternative transient assay to study target genes’ effects on heat and oxidative stress responses.ResultsIn this study, a perennial ryegrass (Loliumperenne L.) mesophyll protoplast-based assay was established to effectively over- or down-regulate target genes. The relative expression levels of the target genes could be quantified using RT-qPCR, and the effects of heat and H2O2-induced oxidative stress on protoplasts’ viability could be quantitatively measured. The practicality of the assay was demonstrated by identifying the potential thermos-sensor genes LpTT3.1/LpTT3.2 in ryegrass that over-expressing these genes significantly altered protoplasts’ viability rates after heat stress.ConclusionThis protoplast-based rapid stress regulatory gene identification assay was briefed as ‘PRIDA’ that will complement the stable genetic transformation studies to rapidly identify candidate stress-regulatory genes in perennial ryegrass and other grass species.