Detection of 210 kDa receptor protein for a leaf-movement factor by using novel photoaffinity probes

Circadian rhythmic plant leaf-movement, called nyctinasty, is controlled by a time-course change in the internal concentration of the leaf-movement factor in the plant body. We revealed that specific binding proteins (210 and 180 kDa) for the leaf-movement factor, potassium lespedezate (1), are contained in the plasma membrane of the plant motor cell by using novel synthetic photoaffinity probes. These proteins are localized on the motor cell in the plant body, and would be potential receptors for the leaf-movement factor to control the leaf-movement. Our study is a rare successful result of the detection of membrane receptors by using a synthetic photoaffinity probe designed on a biologically active natural product. And these results also advance a guideline for probe design towards successful photoaffinity labeling.     

Chemistry and Biology of Plant Leaf Movements

The leaves of Mimosa pudica L. are well known for their rapid movement when touched. Recently, we were able to isolate an excitatory substance in small quantities from this plant, which consists of three different components (potassium L-malate, magnesium trans-aconitate, and dimethylammonium salt). Many plants close their leaves in the evening, as if to sleep, and open them early in the morning (nyctinastic leaf movement). This circadian rhythm is known to be controlled by the biological clock of such plants. Extensive studies on other nyctinastic plants led to the isolation of a variety of leaf-opening substances (LOSs) and leaf-closing substances (LCSs). Based on our experiments on these bioactive substances, we found that the circadian rhythmic leaf movement of these plants is initiated by the regulated balance of LOSs and LCSs. The balance of concentration between the two leaf-movement factors (LMFs) is inversed during the day. The glycoside-type LMF is hydrolyzed with beta-glucosidase, the activity of which is regulated by the biological clock. The circadian rhythm observed in the leaf movement is introduced by activation of beta-glucosidase regulated by the biological clock.     

Synthesis of a novel bioactive photoaffinity probe based on a leaf-movement factor with potential high binding affinity to its receptor molecule

Nyctinastic leaf-movement is induced by the binding of the leaf-movement factor with a motor cell located in the pulvini of the plant. Some receptors of the leaf-movement factor should be involved in this biological event. We developed a novel photoaffinity probe (1) for the detection of receptor molecules. Probe compound 1 bears a photolabeling group on the 2′-position of the glycon moiety, which is near the potential binding site of the molecule with its receptor. This molecular design would be effective for the improvement of photolabeling yield.     

Nanometer-scale direct observation of the receptor for the leaf-movement factor in plant cell by a novel TEM probe

Electron microscopy is a useful method for observing localization of some proteins in a cell. In this Letter, we report a nanometer-scale direct observation of the receptor for a bioactive substance of small molecular weight by using TEM (transmission electron microscopy). We developed a novel TEM probe compound (1) that is a modified leaf-movement factor with benzophenone as a photoaffinity group and FITC as an antigen that is recognized by a nano-gold bound anti-FITC antibody. By using probe 1, we revealed that the receptor for the leaf-movement factor of Cassia mimosoides is localized in the plasma membrane of the plant motor cell.     

Fluorescence studies on nyctinasty using fluorescence labeled cis-p-coumaroylagmatine, a leaf-opening substance of Albizzia plants: existence of genus-specific receptor for leaf-movement factor

We developed fluorescent probes (1 and 2) based on the structure of cis-p-coumaroylagmatine (3), a leaf-opening substance of Albizzia julibrissin Durazz. These probes were effective for the leaf-opening of A. julibrissin, and specifically bind to the motor cell of this plant. Moreover, binding of the fluorescent probe was specific to the plant motor cell contained in the plants belonging to the Albizzia genus. These results showed that the binding of a probe compound with a motor cell is specific to the plant genus and suggested that the genus-specific receptor molecule for the leaf-movement factor on a motor cell would be involved in nyctinasty.     

Fluorescence studies on nyctinasty which suggest the existence of genus-specific receptors for leaf-movement factor

Periodic leaf-movement of legumes is called nyctinasty and has been known since the age of Alexander the Great. We found that nyctinasty is controlled by a periodic change of the internal concentration of leaf-opening and leaf-closing substances in the plant body. Now, we have developed novel fluorescent probes (1) based on the structure of cis-p-coumaroylagmatine (3), which was isolated as a leaf-opening substance of Albizzia juribrissin Durazz. Binding experiments using probe 1 showed that Albizza plants have receptors for a leaf-opening substance in their motor cells. By using probes 1 we then found that genus-specific receptors are involved in nyctinasty.     

Phyllurine, leaf-opening substance of a nyctinastic plant, Phyllanthus urinaria L.

We have isolated phyllurine (1) as the leaf-opening substance of Phyllanthus urinaria L. that keeps the Phyllanthus leaves open. 1 was quite effective for the leaf-opening of Phyllanthus urinaria L. at 2.5 ×10⁻⁵ M, but not effective for other nyctinastic plants even at 1 × 10⁻⁴ M. The leaf-closing substance of this plant has already been identified as phyllanthurinolactone (2). Thus, the leaf-movement of Phyllanthus urinaria L. is proposed to be controlled by the interaction between 1 and 2, similar to the case of Lespedza cuneata G. Don.     

A Study of Nutrient Recycling in a Plant - Soil System Using NAA

To understand how nutrients are recycled in a particular soil - plant system, we have analyzed the kinetics of the elements Na, Mg, K, Ca, Mn, and Br throughout the life cycle of the plant. The plant sample was collected at 6 different stages of its growth. The effect of V (100 µM) on the element recycling system was also analyzed. The amount of the elements in each tissue of the plant, root, stem, petiole, leaf, and seed, were then determined by neutron activation analysis. The total elemental uptake rapidly increased when the plant developed from the juvenile to the adult phase. More than half of the Mg and Mn in cotyledon was transported to the younger leaves before the cotyledon was shed. From the pattern of movement of each element in plant tissue we have constructed an element recycling model in the soil-plant system. Based on the model, it was found that the amount of K to be a limiting factor for the recycling activity in the system.     

Turgorins,Hormones of the Endogeneous Daily Rhythms of Higher Organized Plants—Detection,Isolation, Structure,Synthesis, and Activity

Probably the most fascinating biological phenomenon is the movement which is so impressively observed in higher organized plants when, e.g., in a sort of “quick retreat”, the sensitive plant apparently vanishes on touching, or when all the pinnules of an acacia fold together pairwise at dusk, as though the whole tree were going to “sleep”. Such plants and trees do not possess muscles for the movement of their organs; instead of the contraction of a “still primitive” actomyosin system in this movement the hydrostatic internal pressure of the vacuoles—the turgor—in the parenchymal cells of the motile organs is often drastically and unilaterally reduced. After preception of an external stimulus there follows a gradual or sudden change in the semipermeability of the boundry plasma layers, and, mediated by a stimulus conduction, also of all the symplasts of a multicellular plant organism. The phytohormones which form the molecular basis of the physiology of movement discussed here have now been isolated and structurally elucidated. The chemical agonists of phytodynamics, referred to as turgorins, elicit leaf movement; moreover, they very probably also regulate—perhaps together with other phytohormones—the mechanism of change of aperture in stomatal transpiration. Hence, the turgorins facilitate not only a regulation of temperature, but even their own transport in the sap of the whole plant body.     

Acute and sub-acute toxicological evaluation of the alcoholic leaf and root extracts of Clerodendrum infortunatum L.

Leaf and root extracts of Clerodendrum infortunatum L. have been reported to show anthelmintic efficacy on a cestode parasite Raillietina tetragona. Its leaf showed no toxicity at 1000 mg/kg body weight but root toxicity study was not known. Therefore, our study is to test both leaf and root extracts at 2000 and 3000 mg/kg body weight concentration given orally for 15 days in four groups of Swiss albino mice, keeping another set as control (without plant extract). Weight and behaviour of mice were recorded daily. Feeding, movement pattern were normal in all treatments as that of control. Though body weight increase, there was no change in the relative organ weight. Biochemical and haematological studies revealed no significant change from control and no alteration in histopathological study of liver and kidney from that of control. The plant extracts thus shown to be safe for consumption.     

Water movement in a plant sample by neutron beam analysis as well as positron emission tracer imaging system

We present water imaging of a plant sample both by neutron beam and positron emission tracer imaging system (PETIS). The former method provided static water profile in a plant sample as well as that in the vicinity of a root imbedded in soil. Not only X-ray film but also CT method using a cooled CCD camera is presented. Through non-destructive water image in an X-ray film, root development as well as 2-dimensional water movement toward the root was analyzed. Spatial water image was constructed from 180 CT projection images, taken at an interval of one degree while rotating the sample, through a CCD camera. In the case of a soybean root, there was a water gradient toward a root in soil and gave minimum value at about 1 mm far from the surface of a root. The water absorbing part in a root was gradually shifted downward with the root development. We also present real time water movement by PETIS, where water was labeled with a positron emitting nuclide, ¹⁵O. The transportation of ¹⁵O-water within a plant was relatively slow and water uptake was observed only at the lowest internode, between a root and the first leaf, during 20-minute measurement.     

Antioxidant and antibacterial activity of silver nanoparticles biosynthesized using Chenopodium murale leaf extract

Silver is known for its antimicrobial effects and silver nanoparticles are gaining their importance due to their antimicrobial activities. The aims of the current study were to use plant extract for the biosynthesis of silver nanoparticles and to evaluate their antibacterial and antioxidant activity in vitro. The results indicated that silver nanoparticles (AgNPs) can be synthesized in a simple method using Chenopodium murale leaf extract. The TEM analysis showed that the sizes of the synthesized AgNps ranged from 30 to 50 nm. The essential oil of C. murale leaf extract was formed mainly of α-Terpinene, (Z)-Ascaridole and cis-Ascaridole. The total phenolic compounds and total flavonides were higher in AgNPs-containing plant extract compared to the plant extract. AgNPs-containing leaf extract showed a higher antioxidant and antimicrobial activity compared to C. murale leaf extract alone or silver nitrate. It could be concluded that C. murale leaf extract can be used effectively in the production of potential antioxidant and antimicrobial AgNPs for commercial application.     

Identification of water storage tissue in the stem of cowpea plant (Vigna unguliculata Walp) by neutron radiography

Cowpea (Vigna unguliculata Walp) is considered one of the most drought resistant species among the pulse crops. It was suggested that in the lower part of the stem, parenchymatous tissue for storing water has been developed for the function of drought resistance. However, such tissue has not been identified yet. In order to identify the water storing tissue in the stem of cowpea plant, the authors performed neutron radiography, which provides a non-destructive image of water distribution pattern in a plant. Common bean plant and soybean plant were used as references. Comparing the neutron radiograph for the stems of the plants, i.e., cowpea, common bean and soybean plants, the parenchymatous tissue with water storing function was distinguished in the intermode between primary leaf and the first trifoliate leaf specifically in cowpea plant.     

Application of positron emission tomography and 2-[18F]fluoro-2-deoxy-d-glucose for visualization and quantification of solute transport in plant tissues

The aim of the presented work was to evaluate the uptake and distribution of 2-fluoro-2-deoxy-d-glucose spiked with ¹⁸F (2-[¹⁸F]FDG) in tissues of tobacco plants (Nicotiana tabacum L.) by positron emission tomography and multivariate data analysis after the immersion of the petiole of excised leaf or root of a tobacco plant in a glucose solution. From individual experiments it was found that increasing glucose concentration (c _glu) in the applied solution resulted in significantly higher 2-[¹⁸F]FDG diffusion and translocation within the leaf parenchyma. More than a four times increase of the 2-[¹⁸F]FDG translocation into the aboveground parts of the tobacco plant in case of the root immersion in solution with 100-times higher c _glu in comparison with the control (c _glu = 0.00762 mg cm^?3) was determined. These facts were not confirmed only visually on basis of the obtained 3D images, but also by the increasing coincidence transfer factor (TF_c) values defined by the ratio of the number of analyzed coincidences in the non-immersed parts of leaf or plant to coincidences in leaf petiole or root immersed in the solution. Cluster and principal component analysis suggest that the 2-[¹⁸F]FDG uptake by the petiole of excised leaf and root system was realized by different mechanisms; also, the 3D image quality is influenced by the initial radioactivity of the applied solution.     

青海柴达木地区植物白刺叶中微量元素特征

青海柴达木地区分布有丰富的白刺植物资源。利用220FS原子吸收光谱仪分析了该地区三种白刺叶片的Cu,Zn,Fe,Mn,Cr,Ni,Cd等微量元素。结果表明，铬、 锰含量显著提高是青海柴达木地区植物白刺叶资源微量元素特征。这为柴达木地区白刺叶资源的开发利用提供科学依据。     

Transfer of uranium and thorium from soil to different parts of medicinal plants using SSNTD

The uptake of ²³⁸U and ²³²Th in different parts of some selected plants used in traditional treatment of hypertension and diabetes in south-eastern Morocco (Errachidia area) has been studied using two different types of solid state nuclear track detectors (SSNTDs) LR-115 type II and CR-39. Plant uptake of radionuclides is one of many vectors for introduction of contaminants into the human food chain. Thus, it is critical to understand soil–plant relationships that control nuclide bioavailability. Soil concentrations of uranium ranged from 6.10 to 11.62 ppm, with a mean of 7.90 ppm. Soil concentrations of thorium ranged from 2.70 to 4.80 ppm, with a mean of 3.41 ppm. Mean uranium specific activities were 8.38 Bq kg⁻¹ in root tissue, 5 Bq kg⁻¹ in stem tissue and 6.02 Bq kg⁻¹ in leaf tissue. Mean thorium specific activities were 2.53 Bq kg⁻¹ in root tissue, 1.64 Bq kg⁻¹ in stem tissue and 1.96 Bq kg⁻¹ in leaf tissue. The transfer factors of ²³⁸U and ²³²Th from soil to different parts (root, stem, leaf, seed and fruit) of studied plant samples have been investigated. The transfer factors obtained for root plants were markedly higher than those for leaf, stem, fruit and seed plants. Soil-to-plant transfer factor (TF) is one of the most important parameters to be used in transfer models for predicting the concentration of radionuclides in agricultural crops and for estimating dose impacts to man. This study of uranium and thorium uptake in plants used in traditional medicine is also significant as far as the health hazard effects of uranium and thorium in human being are concerned.     

Secure and Sustainable Sourcing of Plant Tissues for the Exhaustive Exploration of Their Chemodiversity

The main challenge of plant chemical diversity exploration is how to develop tools to study exhaustively plant tissues. Their sustainable sourcing is a limitation as bioguided strategies and dereplication need quite large amounts of plant material. We examine if alternative solutions could overcome these difficulties by obtaining a secure, sustainable, and scalable source of tissues able to biosynthesize an array of metabolites. As this approach would be as independent of the botanical origin as possible, we chose eight plant species from different families. We applied a four steps culture establishment procedure, monitoring targeted compounds through mass spectrometry-based analytical methods. We also characterized the capacities of leaf explants in culture to produce diverse secondary metabolites. In vitro cultures were successfully established for six species with leaf explants still producing a diversity of compounds after the culture establishment procedure. Furthermore, explants from leaves of axenic plantlets were also analyzed. The detection of marker compounds was confirmed after six days in culture for all tested species. Our results show that the first stage of this approach aiming at easing exploration of plant chemodiversity was completed, and leaf tissues could offer an interesting alternative providing a constant source of natural compounds.     

Comparative investigation on chemical constituents of flower bud,stem and leaf of Lonicera japonica Thunb. by HPLC-DAD-ESI-MS/MS n and GC-MS

In approaches of HPLC coupled with diode array detector tandem multiple mass spectrometry (HPLC-DAD-ESI-MS/MS ⁿ ) and GC-MS techniques, efficient comparative methods were developed to profile the chemical constituents in flower bud, leaf and stem parts of Lonicera japonica Thunb. (LJT). 22 polar compounds of various chemical structures and 19 volatile or semi-volatile compounds were tentatively identified from aerial parts of the plant. A number of common composition groups in three separate parts of the plant were proposed, including phenolic acids, flavonoids, iridoids, alkanes, olefins and sterols, the leaf showing higher similarity with the flower bud part via their chemical constituents. Considering the fact that the leaf part has a lot of similar components with the flower bud, this study indicates the leaf part of LJT can be expected to be used as an alternative medicinal resource to the flower bud and stem of the plant.     

Determination of heavy metal concentrations in plants exposed to different degrees of pollution using ICP-AES

Plant samples (Plantago lanceolata – narrow leaf plantain and Cichorium endiviae – endive) were collected in the surroundings of heavy metal emission sources and in other less contaminated areas. After digestion in a closed microwave system using HNO₃, the concentrations of Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Pb, and Zn were determined using ICP-AES. Detection limits for all the elements of interest are given. Differences in heavy metal uptake rate between both plant species were observed. The uptake is more intensive for endive than for narrow leaf plantain. High concentrations of some heavy metals were determined in the unwashed plant samples as a result of exposure to aerosols. Tukeys statistical test was used to confirm the discrepancy of Cr concentration in plant samples from various areas. Washing the leaves with water was found to remove a large amount of water-soluble aerosols.