Multiple amino acid transporters as carriers load L-valine-phenazine-1-carboxylic acid conjugate into Ricinus sieve tubes for the phloem translocation.

Some transporters play important roles in the uptake and acropetal xylem translocation of vectorized agrochemicals. However, it is poorly understood the basipetally phloem-loading functions of transporters toward vectorized agrochemicals. Here, L-Val-PCA (L-valine-phenazine-1-carboxylic acid conjugate) uptake was demonstrated carrier-mediated. RcAAP2, RcANT7, and RcLHT1 showed a similarly up-regulated expression pattern from 62 transporter coding genes in Ricinus at 1 h after L-Val or L-Val-PCA treatment. Subcellular localization revealed that fusion RcAAP2-eGFP, RcANT7-eGFP and RcLHT1-eGFP proteins were expressed in the plasma membrane of mesophyll and phloem cells. Yeast assays found that RcAAP2, RcANT7, and RcLHT1 facilitated L-Val-PCA uptake. To further demonstrate the phloem-loading functions, using vacuum infiltration strategy, an Agrobacterium-mediated RNA interference (RNAi) protocol was constructed in seedlings. HPLC detection indicated that L-Val-PCA phloem sap concentrations were significantly decreased 54.5 %, 27.6 %, and 41.6 % after silencing for 72 h and increased 48.3 %, 52.6 %, and 52.4 % after overexpression, respectively. In conclusion, the plasma membrane-located RcAAP2, RcANT7, and RcLHT1 can loaded L-Val-PCA into Ricinus sieve tubes for the phloem translocation, which may aid in the utilization of transporters and molecular design of phloem-mobile fungicides target root or vascular pathogens.

The translocation and fractionation of rare earth elements (REEs) via the phloem in Phytolacca americana L.

Rare earth elements (REEs) are considered to be emerging contaminants due to their widespread use and lack of recycling. Phytolacca americana L. has great potential for REEs phytoextraction. Our understanding of REEs in P. americana focuses mostly on root absorption and xylem translocation, but the role of phloem translocation has received little attention. In this research, the translocation and fractionation of REEs from phloem to organs in P. americana were investigated. In addition, the effect of organic acids in the REEs translocation via phloem exudates was also examined. The results showed that REEs could transport bidirectionally via the phloem, and 86% of REEs exported from old leaves could move downwards to the root, whereas only 14% of them transported upwards to the young leaves. Heavy rare earth elements (HREEs) enrichment was found in the REEs fractionation processes both from phloem to leaf and from stem to root, indicating that HREEs were preferentially transferred not only down to roots, but also up to the young leaves. The concentration of oxalic acid in phloem exudates was much higher than other organic acids. 94.7% oxalic acid in phloem exudates was preferred to combine with REEs, especially HREEs. Additionally, the concentrations of HREEs had a high positive correlation with oxalic acid in phloem exudates, which demonstrated oxalic acid may play a significant role in the long-distance transport of HREEs in phloem. In conclusion, HREEs have higher translocation ability than light rare earth elements (LREEs) in both xylem and phloem of P. americana. As far as we know, this is the first report focused on the phloem translocation and redistribution of REEs in P. americana, which provides a valuable understanding of the mechanism for phytoremediation of REEs contaminated soils.

Subunit association, and thermal and chemical unfolding of Cucurbitaceae phloem exudate lectins. A review.

Detailed characterization of protein (un)folding intermediates is crucial for understanding the (un)folding pathway, aggregation, stability and their functional properties. In recent years, stress-inducible lectins are being investigated with much interest. In plants phloem proteins PP1 and PP2 are major components of the phloem fluid. While PP1 is a structural protein, PP2 exhibits lectin activity, and was proposed to play key roles in wound sealing, anti-pathogenic activity, and transportation of various molecules including RNA within the plant. Cucurbitaceae fruits contain high concentrations of PP2 lectins, which recognize chitooligosaccharides with high specificity. Although the presence of PP2 lectins in the phloem exudate of Cucurbitaceae species was documented over 40 years ago, so far only a few proteins from this family have been purified and characterized in detail. This review summarizes the results of biophysical studies aimed at investigating the oligomeric status of these lectins, their thermal stability, structural perturbations caused by changes in pH and addition of chaotropic agents and characterization of intermediates observed in the unfolding process. The implications of these results in the functional roles played by PP2 type lectins in their native environment are discussed. Finally, perspectives for future biophysical research on these proteins are given.

An Exploration of the Effect of the Kleier Model and Carrier-Mediated Theory to Design Phloem-Mobile Pesticides Based on Researching the N-Alkylated Derivatives of Phenazine-1-Carboxylic Acid-Glycine.

The Kleier model and Carrier-mediated theory are effective for molecularly designing pesticides with phloem mobility. However, the single Kleier model or Carrier-mediated theory cannot achieve a reliable explanation of the phloem mobility of all exogenous substances. A detailed investigation of the two models and the scope of their applications can provide a more accurate and highly efficient basis for the guidance of the design and development of phloem-mobile pesticides. In the present paper, a strategy using active ingredient-amino acid conjugates as mode compounds is developed based on Carrier-mediated theory. An N-alkylated amino acid is used to improve the pesticide's physicochemical properties following the Kleier model, thus allowing the conjugates to fall on the predicted and more accessible transportation region of phloem. Moreover, the influence of this movement on phloem is inspected by the Kleier model and Carrier-mediated theory. To verify this strategy, a series of N-alkylated phenazine-1-carboxylic acid-glycine compounds (PCA-Gly) were designed and synthesized. The results related to the castor bean seeds (R. communis L.) indicated that all the target compounds (4a−4f) had phloem mobility. The capacity for phloem mobility shows that N-alkylated glycine containing small substituents can significantly improve PCA phloem mobility, such as 4c(i-C3H7-N) > 4a(CH3-N) ≈ 4b(C2H5-N) > 4d (t-C4H9-N) > PCA-Gly > 4e(C6H5-N) > 4f(CH2COOH-N), with an oil−water partition coefficient between 1.2~2.5. In particular, compounds 4a(CH3-N), 4b(C2H5-N), and 4c(i-C3H7-N) present better phloem mobility, with the average concentrations in phloem sap of 14.62 µΜ, 13.98 µΜ, and 17.63 µΜ in the first 5 h, which are 8 to 10 times higher than PCA-Gly (1.71 µΜ). The results reveal that the Kleier model and Carrier-mediated theory play a guiding role in the design of phloem-mobile pesticides. However, the single Kleier model or Carrier-mediated theory are not entirely accurate. Still, there is a synergism between Carrier-mediated theory and the Kleier model for promoting the phloem transport of exogenous compounds. Therefore, we suggest the introduction of endogenous plant compounds as a promoiety to improve the phloem mobility of pesticides through Carrier-mediated theory. It is necessary to consider the improvement of physicochemical properties according to the Kleier model, which can contribute to a scientific theory for developing phloem-mobile pesticides.

Low-pH Molten Globule-Like Form of CIA17, a Chitooligosaccharide-Specific Lectin from the Phloem Exudate of Coccinia indica, Retains Carbohydrate-Binding Ability.

pH-induced changes in the conformation, structural dynamics, and carbohydrate-binding activity of Coccinia indica agglutinin (CIA17), a PP2-type lectin, were investigated employing biophysical approaches. The secondary structure of CIA17 remains nearly unaltered over a wide pH range (2.0-8.5), while the tertiary structure of the protein exhibits considerable changes. A decrease in the fluorescence intensity and excited-state lifetime at low pH indicated perturbation in the local conformation (near Trp residues) of CIA17, which was further supported by enhancement in the Trp accessibility toward charged quenchers under acidic conditions. Fluorescence correlation spectroscopic studies indicated that at pH 2.0, CIA17 exists as a monomer over the concentration range of 10-200 nM and forms dimers at higher concentrations (KD ∼ 387 nM) but could not form higher oligomers even at ∼150-fold higher concentrations, unlike under native conditions at pH 7.4. Thermal unfolding of the low pH intermediate involves two distinct steps: dissociation of a dimer to a monomer, followed by the unfolding of the monomer. These results strongly suggest that the acid-induced unfolding pathway of CIA17 involves the formation of a monomeric molten globule-like intermediate, which retains appreciable carbohydrate-binding ability. These observations are of great physiological significance since the PP2 proteins are involved in plant defense responses.

Extraction of cucumber phloem sap based on the capillary-air pressure principle.

Changes in the substances in phloem sap can effectively reflect the nutritional status of cucumber plants during their growth. Because of the limitations of the time-consuming and complex operations of existing phloem sap extraction methods, the authors proposed a new extraction method based on the capillary-air pressure principle and designed a new sap sampling device. To examine the feasibility of the new sampling device, sap sampled from the same plant with the new method and the common EDTA method was analyzed by gas-phase mass spectrometry. The data showed that the number of substances in the sap sampled using capillary-air pressure was higher than that observed using the EDTA method. The concentration of substances sampled using capillary-air pressure was much higher than that observed using EDTA.

Lignin concentrations in phloem and outer bark are not associated with resistance to mountain pine beetle among high elevation pines.

A key component in understanding plant-insect interactions is the nature of host defenses. Research on defense traits among Pinus species has focused on specialized metabolites and axial resin ducts, but the role of lignin in defense within diverse systems is unclear. We investigated lignin levels in the outer bark and phloem of P. longaeva, P. balfouriana, and P. flexilis; tree species growing at high elevations in the western United States known to differ in susceptibility to mountain pine beetle (Dendroctonus ponderosae; MPB). Pinus longaeva and P. balfouriana are attacked by MPB less frequently than P. flexilis, and MPB brood production in P. longaeva is limited. Because greater lignification of feeding tissues has been shown to provide defense against bark beetles in related genera, such as Picea, we hypothesized that P. longaeva and P. balfouriana would have greater lignin concentrations than P. flexilis. Contrary to expectations, we found that the more MPB-susceptible P. flexilis had greater phloem lignin levels than the less susceptible P. longaeva and P. balfouriana. No differences in outer bark lignin levels among the species were found. We conclude that lignification in Pinus phloem and outer bark is likely not adaptive as a physical defense against MPB.

Improving pesticide uptake modeling and management in potatoes: A simple and approximate phloem-adjusted model.

To evaluate the impact of the phloem flux on the pesticide uptake process in potatoes, this study developed a phloem-adjusted model based on the classic model that focuses mainly on the diffusion process. To achieve high-throughput simulations, we introduced an approximate method to convert the phloem flux transport process into a simple specific uptake rate of pesticides. In comparison to the classic model (non-phloem model), the phloem-adjusted model generated higher pesticide concentrations and bioconcentration factors (BCFs) in potatoes, owing to the additional pesticide uptake route introduced to the adjusted model. However, the simulation, which was conducted for 740 pesticides, indicated that for most pesticides, the phloem flux route did not contribute a significant portion of the pesticide uptake to potato tubers compared with the soil diffusion route. This was further characterized, using the differential factor (DF), to evaluate the difference in the simulated results between the proposed model and classic models. The largest DF (~0.11) was obtained for pesticides with moderate lipophilicity (i.e., log KOW of 3.0), indicating that only a difference of 10% was generated between the two models. The 10% increase in pesticide concentration (or BCFs) in potatoes, simulated by the phloem-adjusted model, was within the acceptable uncertainty interval of the classic model, thus confirming the validity of using the classic model to predict the pesticide uptake process in potato tubers. However, we found that the negligibility of the phloem flux route was not merely due to hydrophobicity (i.e., hypothesis of the classic model), but was related to the i) plant physiology of potatoes, ii) lipophilicity of a pesticide, and iii) the diffusivity of a pesticide in water. Although future studies on pesticide concentrations in phloem sap and the dynamic growth of potatoes need to be undertaken, the model developed in this study reveals a more comprehensive pesticide uptake process in potatoes, which can promote the understanding of the pesticide uptake mechanism in potatoes.

Collection and Analysis of Phloem Lipids.

The plant phloem is a long-distance conduit for the transport of assimilates but also of mobile developmental and stress signals. These signals can be sugars, metabolites, amino acids, peptides, proteins, microRNA, or mRNA. Yet small lipophilic molecules such as oxylipins and, more recently, phospholipids have emerged as possible long-distance signals as well. Analysis of phloem (phospho)lipids, however, requires enrichment, purification, and sensitive analysis. This chapter describes the EDTA-facilitated approach of phloem exudate collection, phase partitioning against chloroform-methanol for lipid separation and enrichment, and analysis/identification of phloem lipids using LC-MS with multiplexed collision induced dissociation (CID).

Predator Performance and Fitness Is Dictated by Herbivore Prey Type Plus Indirect Effects of their Host Plant.

Animals, including herbivores and predators, use diet-mixing to balance their macro- and micronutrient intake. Recent work demonstrated that lady beetles fed only pea aphids from fava beans had reduced fitness caused by a deficiency of dietary sterols. However, beetles redressed this deficit by eating fava bean leaves. In the current study we used Coccinella septempunctata as a model to test the hypotheses that pea aphids are a poor sterol resource independent of their host plant, and that fava beans produce low quality prey regardless of aphid species. Additionally, we tested the reproductive rescue capacity of alfalfa and barley foliage compared to fava, and profiled the sterols of phloem exudates, foliage, and aphids reared on these different hosts. Beetle fecundity and egg viability was significantly better when provided pea aphids reared on alfalfa (compared to fava beans) and green peach aphids reared on fava plants. Alfalfa and barley leaves were not consumed by beetles and did not support beetle reproduction. The sterol profile of aphids largely reflected their host plant phloem. However, green peach aphids from fava acquired 125-times more sterol than pea aphids from fava. Our findings show how the sterol content of different host-plants can affect the third trophic level. Our results suggest that 1) prey quality varies depending on prey species, even when they occur on the same plant, 2) plant species can mediate prey quality, 3) host plant-mediated effects on prey quality partially drive omnivory, and 4) diet-mixing benefits growth and reproduction by redressing micronutrient deficits.

Analysis of the functional group composition of the spruce and birch phloem lignin.

In this article, the functional group composition of the spruce (Pícea ábies) and birch (Bétula péndula) phloem lignin is described. The features of the chemical structure were studied by analyzing dioxane lignin using the elemental analysis, UV-Vis, FT-IR, and 1D NMR spectroscopy. For comparison, samples of xylem dioxane lignin isolated from the corresponding wood species were also analyzed. FT-IR spectroscopy data suggest that the lignins of birch phloem and xylem are similar in chemical structure. However, there are differences in absorption bands in the spectra of spruce dioxane lignin, which indicate the opposite. Quantitative analysis of the functional group composition was performed using 13C and 31P NMR data. It was found that free phenolic hydroxyl groups of catechol and p-hydroxyphenyl types are dominated in the composition of spruce phloem lignin. Birch phloem lignin has a qualitative and quantitative composition of functional groups characteristic of hardwood lignins. However, the content of G-units is greater than S-units, in contrast to the birch xylem lignin, where S-units predominate. The revealed differences are relevant from the point of view of plant physiology. The practical significance of the study is connected with understanding the reactivity of lignins when considering the chemical processing of tree bark.

Localization of (+)-Catechin in Picea abies Phloem: Responses to Wounding and Fungal Inoculation.

To understand the positional and temporal defense mechanisms of coniferous tree bark at the tissue and cellular levels, the phloem topochemistry and structural properties were examined after artificially induced bark defense reactions. Wounding and fungal inoculation with Endoconidiophora polonica of spruce bark were carried out, and phloem tissues were frequently collected to follow the temporal and spatial progress of chemical and structural responses. The changes in (+)-catechin, (-)-epicatechin, stilbene glucoside, and resin acid distribution, and accumulation patterns within the phloem, were mapped using time-of-flight secondary ion mass spectrometry (cryo-ToF-SIMS), alongside detailed structural (LM, TEM, SEM) and quantitative chemical microanalyses of the tissues. Our results show that axial phloem parenchyma cells of Norway spruce contain (+)-catechins, the amount of which locally increases in response to fungal inoculation. The preformed, constitutive distribution and accumulation patterns of (+)-catechins closely follow those of stilbene glucosides. Phloem phenolics are not translocated but form a layered defense barrier with oleoresin compounds in response to pathogen attack. Our results suggest that axial phloem parenchyma cells are the primary location for (+)-catechin storage and synthesis in Norway spruce phloem. Chemical mapping of bark defensive metabolites by cryo-ToF-SIMS, in addition to structural and chemical microanalyses of the defense reactions, can provide novel information on the local amplitudes and localizations of chemical and structural defense mechanisms and pathogen-host interactions of trees.

Phloem sap in Cretaceous ambers as abundant double emulsions preserving organic and inorganic residues.

Fossilized remains preserved in amber provide abundant data on the paleobiota surrounding the resin-producing plants, but relatively scarcer information about the resinous sources themselves. Here, dark pseudoinclusions in kidney-shaped amber pieces from the Early Cretaceous (Albian) amber from Spain are studied. This type of fossilized remain, abundant in Cretaceous ambers, was first interpreted as fossilized vacuole-bearing microorganisms, but later regarded as artifactual and probably secreted by the resinous trees, although their origin remained unclear. Using complementary microscopy (light, electron, confocal), spectroscopy (infrared, micro-Raman), mass spectrometry and elemental analysis techniques, we demonstrate that the pseudoinclusions correspond to droplets of phloem sap containing amber spheroids and preserving both organic and inorganic residues consistent with degraded components from the original sap. The amber pieces containing pseudoinclusions are fossilized, resin-in-sap-in-resin double emulsions, showing banding patterns with differential content of resin-in-sap emulsion droplets. Our findings represent the first time fossilized phloem sap, 105 million years old, has been recognized and characterized, and open new lines of paleontological research with taxonomic, taphonomic, physiological and ecological implications.

Improving the extraction and purification of leaf and phloem sugars for oxygen isotope analyses.

RATIONALE: The oxygen isotopic composition (here shown as the δ18 O value) of soluble sugars in leaves and phloem tissue holds valuable information about plant functions in response to climatic changes. However, δ18 O analysis of sugars is prone to error, and thoroughly tested methods are lacking. METHODS: We performed three experiments to test if sample preparation modifies the δ18 O values of sugars. In experiment 1, we tested the effects of oven-drying versus freeze-drying, whereas in experiment 2 we focused on the extraction and purification of leaf sugars. In experiment 3, we investigated the exudation and purification of twig phloem sugars as a function of exudation time and different ethylenediaminetetraacetic acid (EDTA) exudation media. RESULTS: Freeze-drying produced more consistent δ18 O values than oven-drying for sucrose but not for phloem sugars. The extraction and purification of leaf sugars can be performed without a significant modification of their δ18 O values; yet the purified leaf and phloem sugars possessed higher δ18 O values than the fraction of water-soluble compounds. Moreover, the exudation time significantly modulated the δ18 O values of phloem sugars, which is probably related to changes in the sugar composition. The addition of EDTA did not improve the determination of the δ18 O values of phloem sugars. CONCLUSIONS: We show that the sample preparation of plant sugars for the reliable determination of δ18 O values requires a strict protocol, which is described in this paper. For phloem sugar, we recommend a maximum exudation time of 1 h to reduce the degradation of sucrose and minimise oxygen isotope exchange reactions between the resulting hexoses and water.

Estimating canopy gross primary production by combining phloem stable isotopes with canopy and mesophyll conductances.

Gross primary production (GPP) is a key component of the forest carbon cycle. However, our knowledge of GPP at the stand scale remains uncertain, because estimates derived from eddy covariance (EC) rely on semi-empirical modelling and the assumptions of the EC technique are sometimes not fully met. We propose using the sap flux/isotope method as an alternative way to estimate canopy GPP, termed GPPiso/SF , at the stand scale and at daily resolution. It is based on canopy conductance inferred from sap flux and intrinsic water-use efficiency estimated from the stable carbon isotope composition of phloem contents. The GPPiso/SF estimate was further corrected for seasonal variations in photosynthetic capacity and mesophyll conductance. We compared our estimate of GPPiso/SF to the GPP derived from PRELES, a model parameterized with EC data. The comparisons were performed in a highly instrumented, boreal Scots pine forest in northern Sweden, including a nitrogen fertilized and a reference plot. The resulting annual and daily GPPiso/SF estimates agreed well with PRELES, in the fertilized plot and the reference plot. We discuss the GPPiso/SF method as an alternative which can be widely applied without terrain restrictions, where the assumptions of EC are not met.

What determines the composition of the phloem sap? Is there any selectivity filter for macromolecules entering the phloem sieve elements?

In view of recent findings, it is still a matter of debate whether the composition of the phloem sap of higher plants is specific and based on a plasmodesmal selectivity filter for macromolecular transport, or whether simply related to size, abundance and half-life of the macromolecules within the phloem sap. A range of reports indicates specific function of phloem-mobile signaling molecules such as the florigen making it indispensable to discriminate specific macromolecules entering the phloem from others which cannot cross this selectivity filter. Nevertheless, several findings have discussed for a non-selective transport via plasmodesmata, or contamination of the phloem sap by degradation products coming from immature still developing young sieve elements undergoing differentiation. Here, we discuss several possibilities, and raise the question how selectivity of the phloem sap composition could be achieved thereby focusing on mobility and dynamics of sucrose transporter mRNA and proteins.

Positron-emitting tracer imaging of fluoride transport and distribution in tea plant.

BACKGROUND: Tea (Camellia sinensis (L.) O. Kuntze) is a hyper-accumulator of fluoride (F). To understand F uptake and distribution in living plants, we visually evaluated the real-time transport of F absorbed by roots and leaves using a positron-emitting (18 F) fluoride tracer and a positron-emitting tracer imaging system. RESULTS: F arrived at an aerial plant part about 1.5 h after absorption by roots, suggesting that tea roots had a retention effect on F, and then was transported upward mainly via the xylem and little via the phloem along the tea stem, but no F was observed in the leaves within the initial 8 h. F absorbed via a cut petiole (leaf 4) was mainly transported downward along the stem within the initial 2 h. Although F was first detected in the top and ipsilateral leaves, it was not detected in tea roots by the end of the monitoring. During the monitoring time, F principally accumulated in the node. CONCLUSION: F uptake by the petiole of excised leaf and root system was realized in different ways. The nodes indicated that they may play pivotal roles in the transport of F in tea plants. © 2020 Society of Chemical Industry.

Sugar, amino acid and inorganic ion profiling of the honeydew from different hemipteran species feeding on Abies alba and Picea abies.

Several hemipteran species feed on the phloem sap of plants and produce large amounts of honeydew that is collected by bees to produce honeydew honey. Therefore, it is important to know whether it is predominantly the hemipteran species or the host plant to influence the honeydew composition. This is particularly relevant for those botanical and zoological species from which the majority of honeydew honey originates. To investigate this issue, honeydew from two Cinara species located on Abies alba as well as from two Cinara and two Physokermes species located on Picea abies were collected. Phloem exudates of the host plants were also analyzed. Honeydew of all species contained different proportions of hexoses, sucrose, melezitose, erlose, and further di- and trisaccharides, whereas the phloem exudates of the host trees contained no trisaccharides. Moreover, the proportions of sugars differed significantly between hemipteran species feeding on the same tree species. Sucrose hydrolysis and oligosaccharide formation was shown in whole-body homogenates of aphids. The type of the produced oligosaccharides in the aphid-extracts correlated with the oligosaccharide composition in the honeydew of the different aphid species. The total contents of amino acids and inorganic ions in the honeydew were much lower than the sugar content. Glutamine and glutamate were predominant amino acids in the honeydew of all six hemipteran species and also in the phloem exudates of both tree species. Potassium was the dominant inorganic ion in all honeydew samples and also in the phloem exudate. Statistical analyses reveal that the sugar composition of honeydew is determined more by the hemipteran species than by the host plant. Consequently, it can be assumed that the sugar composition of honeydew honey is also more influenced by the hemipteran species than by the host tree.

Application of capillary electrophoretic methods for the analysis of plant phloem and xylem saps composition: A review.

Plant vascular tissue is essential for the exchange of water, nutrients, metabolic products, and signals among distant organs in cormophytes. The compositions of phloem and xylem saps are highly dependent on many internal and external factors, and thus their analysis provides a valuable insight into plant physiology, growth, and development as well as nutrition status or presence of biotic or abiotic stresses. Capillary electrophoresis characterized by highly efficient separations and minuscule sample requirements represents a suitable analytical technique for this purpose because the sap constitutes a complex mixture with generally minimal availability. This review aims at providing a comprehensive overview of published capillary electrophoretic methods for the analysis of primary components present in the phloem and xylem saps of higher plants.

Optimal Preparation of Tissue Sections for Light-Microscopic Analysis of Phloem Anatomy.

In order to successfully analyze and describe any plant tissue, the first challenge is preparation of good anatomical slides. The challenge is even greater when the target tissue has heterogeneous characteristics, such as the phloem where soft and stiff tissues occur side by side. The goal of this chapter is to present a detailed protocol containing various techniques for optimal preparation of phloem tissue samples for light microscopic analysis. The process typically involves the steps of fixation, softening, embedding, sectioning, staining, and mounting. The protocol can be applied to make samples of phloem and surrounding tissues of stems and roots, from woody to herbaceous plants.