Xylem and phloem sap protein extraction experiments
It is well known that the xylem and primordial vasculature in higher plants are responsible for the long-distance transport of small nutrient molecules. However, the presence of proteins in these two transport sap types has not been anticipated and the function of these proteins has not been thoroughly investigated. In this chapter, we describe how to extract and purify proteins from xylem and phloem sap for proteomic analysis. The source for this experiment is the "Guide to Plant Proteomics Experiments" [France] H. Tillmant, M. Zivi, C. Damerweil, V. Mitschine, eds.
Operation method
Xylem and phloem sap protein extraction
Materials and Instruments
Phloem sap Move 3.1 Collection of xylem sap For more product details, please visit Aladdin Scientific website.
HCl NaOH Acetone Methanol DTT β-phenylmercaptoethanol
Razor blade Screw cap test tube
Xylem sap belongs to the extracellular space of plants and contains specific proteins whose types and concentrations vary with the state of the plant. Because pure xylem sap is readily available from most plants, it is easy to identify xylem sap proteins using proteomics tools. Preparation of protein samples for one-way electrophoresis (1- DE) or two-way electrophoresis (2- DE) separation takes into account the fact that the protein concentration is very low and contains oligosaccharides (and glycosylated proteins). There are two basic steps in the analysis of xylem sap protein samples by 1- DE or 2- DE, i.e., collection of xylem sap and concentration of proteins.
( 1 ) Collect the sap that naturally flows out of the root side stems (due to root pressure runoff) by cutting the stems of the plant with a razor blade (Figure 4-1). Plant stems can be cut at any height, but in general, the closer the cut is to the base of the stem, the greater the amount of sap that flows out. However, the lotus should be cut to a certain height (about 10 cm) in order to connect it to a tube placed in ice (see Note 1).
( 2 ) Sap collection.
a. Place the remaining stem segments horizontally and attach a tube to the incision. The tube is placed in a container with ice, and the sap is then collected for up to 6 h ( Fig. 4-1C; the container ice needs to be replenished during the sap collection process; see Note 2).
The sap is then collected in a reaction tube placed on ice. b. Alternatively, oozing xylem sap can be collected repeatedly every few minutes with a pipette, and the liquid is collected into a reaction tube placed on ice. 
( 3 ) The cuts are cleaned before collecting the sap to remove the cytoplasm of the cut cells and the sap that flowed directly from the cut phloem (see Note 3) .
( 4 ) Granular material, like soil particles, microbial cells, or residual tissue, may be removed by centrifugation before concentrating the protein.
3.2 Concentration of xylem sap proteins
Xylem sap contains not only proteins, but also carbohydrates (see Note 4 ) and other compounds such as amino acids, salts and polyphenols (in pathogen-infested plants ).
If 1-DE is performed, simple protein precipitation is sufficient, even with ethanol (4 parts 95% ethanol/5% 0.1 mol/L NaAc added to 1 protein sample), although ethanol can also precipitate poly- and oligosaccharides.
If isoelectric focusing is performed, the juice should be partially purified and concentrated with an Amicon filter unit, followed by precipitation with TCA / acetone.
1. Partial purification and concentration of proteins by Amicon Filtration
Concentrate and partially purify xylem sap using an Amicon CentriprepYM-3 Centrifugal Ultrafiltration Concentrator (up to 15 ml, auto switch: 3 kDa) and/or an Amicon Centricon Plus-20 Centrifugal Ultrafiltration Tube (larger volume, auto switch: 10 kDa) filtration unit.
( 1 ) After centrifugation to remove particulate matter, add up to 15 ml (AmiconCentriprepYM-3) or 19 ml (Amicon Centricon Plus-20) of xylem sap to the sample container.
( 2 ) Rotate the Amicon Centriprep YM-3 at 4°C, 3000 g for 75 min until equilibrium is reached (between the inside and outside of the filter collector).
( 3 ) Rotate Amicon Centricon Plus-20, 4000 g, for 15 min at 4°C. Gently pour out the filtrate and add 19 ml of xylem sap to the sample filter cup.
( 4 ) Spin again for 15 min.
( 5 ) Pour out the filtrate gently and repeat the spinning and pouring steps if further concentration is required.
( 6 ) Collect the concentrate (at least 500 μl) directly from the Centriprep YM-3.
( 7 ) Spin the inverted unit at 1000 g for 5 min and collect the concentrate (at least 200 μl) from the Centricon Plus-20.
2. Precipitation of proteins with TCA/acetone
( 1 ) Add 4 portions of 100% acetone solution (containing 12.5% TCA and 0.0875% β-mercaptoethanol) to one portion of protein and mix.
( 2 ) React at -20°C for more than 45 min (or with 80% acetone at 0°C for 1~4 h), then centrifuge for 30 min at maximum speed of centrifuge (without freezing).
( 3 ) Discard the supernatant and rinse the precipitate three times with ice-cold 100% acetone.
( 4 ) Remove the residual liquid and dry the precipitate at room temperature. The precipitate can be dissolved in resuspension buffer (2 mol/L thiourea, 7 mol/L urea, 4% CHAPS, 2% mmol/L DTT) [ 5 ] and used for isoelectric focusing, or stored at -20 °C.
3.3 Collection of phloem sap
Phloem sap is more difficult to collect from most plant bodies than xylem sap. Therefore, the main challenge in phloem sap proteomics is collecting a sufficiently large amount of material. However, different collection methods can be applied depending on the characteristics of different plant species.
For some plant species (e.g., castor, cucurbit, filipendula), small incisions or cutting of the entire plant organ [18] can be used to collect phloem sap. Plants unsuitable for this method of collection can be sampled by EDTA-assisted secretion [ 19~21]. Highly pure phloem sap can be obtained from many plants by the aphid needle technique [ 22 ], but in small quantities.
Established model plants, such as Arabidopsis thaliana or rice, have difficulty in collecting sufficient amounts of sieve tube molecular exudates for proteomic studies. In the case of Arabidopsis, the amount of sample available is insufficient for proteomic methods [ 10], and in the case of rice, only a few high-abundance phloem sap proteins can be identified from planthopper sap exudates [ 6, 23]. Most of the current information on identifying bast peptides comes from cucurbits [ 8], castor [10], and more recently from oilseed rape [ 7].
It is relatively easy to collect phloem sap from these plants. Two versions of the exudation technique are described below.
1 . Version 1
( 1 ) Cut the main stem or petiole of the plant with a razor blade (Figure 4-2).
( 2 ) Puncture the plant body with an injection needle (see Note 5).
2 . Version 2
( 1 ) After cutting, use the broken lotus on one side of the above-ground portion and blot the cut with filter paper (see Note 6 ).
( 2 ) Absorb the first droplets of exudate with filter paper.
The subsequently oozing sap is collected by aspiration into a reaction tube kept in ice (see Note 7).
3.4 Purification of proteins from bast sap
( 1 ) When isolating bast proteins in 1-DE or 2-DE, the high concentration of sugars and organic matter in the transport solution must be taken into account. In addition, plants have mechanisms to defend themselves against cuts, which include protein polymerization [ 24 ]. 
( 2 ) For 1-DE, the bast sample can be spiked directly into 1-DE sample buffer "25", heated, and subjected to gel electrophoresis at room temperature.
( 3 ) For 2-DE, proteins should be purified by precipitation before the sample solution is used for isoelectric focusing electrophoresis.
1. Removal of major proteins 1 (PP1) and 2 (PP2) from the phloem.
The two major phloem proteins, phloem filament protein PP1 and phloem lectin PP2, are present in all dicotyledonous plants and account for more than 50% of all phloem proteins in some plants (e.g., cucurbits).
Under oxidizing conditions, both proteins form insoluble polymers through disulfide bonds [ 26, 27], and they form trailing tails in 1-DE and 2-DE, which degrade resolution. To remove these proteins, acidification followed by neutralization can be used [28]. All of the following steps are done at room temperature.
(1 ) Calibrate the sample solution to pH 2.0 with 2 mol/L HCl.
( 2 ) Neutralize the sample solution to pH 7.5 with 2 mol/L NaOH.
( 3 ) Centrifuge at 15 000 g for 15 min.
( 4 ) Collect the supernatant and remove the large white precipitate containing PP1 and PP2 (see Note 8).
2. Precipitation of all proteins from the sap of the primary bark by acetone/methanol/DTT.
To remove carbohydrates and other interfering components, the proteins can be precipitated by treatment with acetone/methanol/DTT.
(1) Add 3 times the volume of pre-cooled precipitation solution.
( 2 ) Precipitate overnight at -20°C (see Note 9).
( 3 ) Centrifuge at 6800 g at 4°C for 5 min (see Note 10) and discard the supernatant.
( 4 ) Rinse the precipitate twice with 100% pre-cooled acetone and centrifuge at 6800 g at 4°C for 5 min.
( 5 ) Remove the supernatant and allow the precipitate to dry at room temperature for 5-10 min (see Note 11).
( 6 ) The precipitate is dissolved in Sample Buffer for 1-DE and 2-DE (see Note 12).