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

Phloem is an important vascular tissue connecting the aboveground and underground parts of plants. As an important part of the vascular system, phloem plays a role in information transmission and mechanical support together with other parts of the vascular system. Phloem also plays an important role in material transport, such as the transport of carbohydrates, amino acids and other nutrients from source to sink [1,2]. The transport and distribution of nutrients directly affect the growth process, yield and quality of plants [3]. When photosynthetic products are loaded and unloaded in the phloem, the health status of the plant itself can be reflected by detecting changes in the types and concentrations of substances. Leaf tissue was sampled to investigate changes in plant matter and concentration in most experiments. In this study, petiole phloem sap was used instead of leaf tissue to determine the health status of plants by studying the composition and concentration of the phloem sap. This method provides a novel approach to the detection of plant nutrient status.

Petioles in the cucumber are small in diameter, so it was necessary to determine the exact location of the phloem on the petioles to ensure pure phloem sap was obtained. Phloem is distributed in the vascular bundle, and the xylem is surrounded by phloem sieve tubes [4,5]. When the petiole is cut off, the sap carrying photosynthetic products exudes from the phloem sieve tube and forms hemispherical droplets. Because of the low amount of phloem sap, its extraction is time-consuming and laborious, so a new method is proposed to obtain phloem sap rapidly.

Methods

Samples

Cucumber seedlings were planted in a Venlo-type greenhouse at the Key Laboratory of Modern Agricultural Equipment and Technology, Ministry of Education, Jiangsu University, Zhenjiang, China (119°45′E, 32°20′N). Plant nutrient-enriched soil (Zhenjiang Peilei Matrix Technology Development Co., Ltd., Zhenjiang, China) was used for seedling cultivation. After 2 weeks, cucumber seedlings with good growth status were selected and transplanted into pots filled with perlite with a particle diameter of 2–4 mm (Zhenjiang Runzhou Xinpei Horticultural Materials Business Department, Zhenjiang, China). Nutrient solution A consisted of 945 mg/l Ca(NO₃)₂·4H₂O, 79 mg/l KNO₃, 40 mg/l NH₄NO₃ and 7 mg/l Fe-EDTA (13.1%) (an iron ion solution), and solution B consisted of 604 mg/l KNO₃, 170 mg/l KH₂PO₄, 339 mg/l MgSO₄·7H₂O, 1.7 mg/l MnSO₄·4H₂O, 1.45 mg/l ZnSO₄·7H₂O, 2.45 mg/l Na₂B₄O₇·10H₂O, 0.19 mg/l CuSO₄·5H₂O and 0.12 mg/l Na₂MoO₄·2H₂O. Solutions A and B were mixed in equal amounts for use. The nutrient solution was applied at 8 am, and plants were irrigated with water at 12 am and 6 pm every day. The greenhouse temperature ranged from 18 to 35°C. The average natural light duration was 5.13 h, and the average daily irradiation was 13.2 MJ/m².

Capillary–air pressure method

Observing petiole structure to determine sampling position

When the plants grew to the flowering and fruiting stage, the petiole was cut along the circumferential direction to form a 2-mm-thick section. The cut section of the petiole was glued using conductive adhesive to the specimen stage. The metal was applied with sputtering at a thickness of 20 nm. The processed thin section was pasted on the sample platform of the scanning electron microscope and observed and imaged under 15-kV acceleration voltage [6].

Electron microscopy image of the cucumber petiole is shown in Figure 1. It revealed that the vascular bundles were distributed in the protruding edge of the petiole, and the number of edge was positively correlated with the number of vascular bundles. The vascular bundle consists of phloem and xylem. Except for the vascular bundles, most of the interior of the epidermis consists of pith. In Figure 1A, the vascular bundle was outlined with red lines (c, vascular bundle), and the pith was outlined with yellow lines (d, pith). Figure 1B was a Partial enlarged graph of petiole structure, which shows epidermis, vascular bundle (phloem sieve tube and xylem catheter) and pith from top to bottom.

The phloem sap was transported in the phloem sieves, and part of the phloem sieve tube was identified by observing the petiole structure under electron microscopy to determine the position of phloem sap sampling. Finally, the sampling position of the phloem sap was determined in the double phloem of the vascular bundle.

Sampling method based on capillary–air pressure principle

The sucrose content in the phloem sap of cucumber is high. The sap is viscous and easily solidified when exposed to air. The sap often clogs extractors (e.g., syringes and pipettes) and cannot be continuously sampled. The capillary and air compression principle is illustrated in Figure 2. Sap can be directly extracted from cucumber phloem into a capillary glass tube (100 and 0.5 mm; Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) (Figure 2A), which can reduce the contact of sap with air, decrease clogging and improve the sampling efficiency. To store the sap, appropriate air pressure is needed to squeeze it out of the capillary glass tube. The main function of the syringe is to provide the appropriate air pressure to force the sap out of the tube.

Sap sampling

First, the petiole of the seventh leaf from the terminal bud was cut down using a sterilized blade. The section was washed with distilled water, and the first drop of sap was wiped with a defatted cotton ball. Second, the leaf was fixed on the worktable with cotton rope. To collect sap, one end of the capillary glass tube was aligned with the phloem sieve tube, and then the capillary tube was kept perpendicular to the section. Finally, the capillary glass tube containing the sample was placed in the needle of the syringe, the sap was squeezed into a 1.5 ml centrifuge tube by squeezing the air. The centrifuge tube containing the sap was stored in liquid nitrogen. The sampling process is shown in Figure 3.

Capillary–air pressure sampling device

Based on the capillary–air pressure sampling method, a simple sampling device was designed and manufactured (Figures 4 & 5). The detailed components are shown in Figure 4, and the physical device is shown in Figure 5. The sampling device is composed mainly of a capillary glass tube clamping apparatus, an air compressor and a storage component. The clamping part of the capillary glass tube is composed of replacement and locking parts, the air compression part has a miniature air pump and the storage part has a fixed sleeve.

When sampling, the capillary glass tube is aimed at the sieve part of the phloem section. Because the work of the micropneumatic pump causes the internal pressure of the pressure chamber becomes lower, resulting in the internal pressure lower than the external pressure, so the sap flow increases in speed and extracted quickly. The sap is continuously extracted under the negative pressure created at the tail end of the capillary tube. Finally, the sap drops into the centrifuge tube automatically under the action of gravity.

Comparison with EDTA method

Method comparison

Samples extracted using different methods may have different compositions. To examine the feasibility of the new extraction device, the same plants were sampled. The capillary–air pressure and EDTA extraction methods were used [7]. The EDTA method was used to obtain the phloem sap according to the following steps. First, three leaves with petiole were removed from the stem. The seventh leaf from the top bud was taken, ensuring that each was disease-free and the leaf area ranged from 500 to 550 cm². Second, the leaves were immediately placed in a Petri dish containing 15 ml of 20-mM EDTA solution. The cut end of the petiole was immersed in the solution. Third, the leaves were gently folded together with the petiole fully immersed in 20-mM EDTA solution, and the leaves and solution were covered with a black bag for 1 h. Fourth, the leaves were removed from the Petri dish and the petiole was cleaned with distilled water. The cleaned leaves were placed into a Petri dish containing 10 ml of distilled water and left in the dark for 1-1/2 h. Fifth, a 1-ml syringe was used to transfer 1 ml of exudate from the Petri dish to a 2.5-ml centrifuge tube. Sixth, the centrifuge tube containing the exudate was immersed in -80°C liquid nitrogen for storage.

Sample pretreatment & testing

For the HPLC detection method, 30 μl of sap was put into a 10-ml centrifuge tube, 2 ml of 80% acetonitrile was added and the solution was mixed well. Next, an ultrasonic bath instrument (PS40A; Huatai Scientific Instruments Co., Ltd. Jiangsu, China) was used to ultrasonicate the sample in a low-temperature water bath for 30 min. The samples were centrifuged in a table-top high-speed centrifuge (TG-16G; Hunan Kaida Scientific Instruments Co., Ltd., Changsha, China) at 1000 x g and 4°C for 5 min, and the supernatant was then removed. Finally, the samples were filtered using a 0.22-μm organic-phase filtration membrane. After sample pretreatment, HPLC (1290, Agilent Technology Co., Ltd., CA, USA) was used to detect the samples.

For the GC-MS detection method, 30 μl of sap was taken from each sample, and 170 μl of deionized water and 800 μl of methanol acetonitrile (1:1 v/v) were then added to the sap. A 53-kHz ultrasonic bath instrument (SK5200GT; Shanghai Kedao Ultrasonic Instrument Co., Ltd. Shanghai, China) was used to ultrasonicate the sample in an ice bath for 1 h, and the samples were then incubated in a low-temperature refrigerator at -20°C for 1 h to precipitate the protein. The samples were centrifuged in a table-top high-speed refrigerated centrifuge (Neofuge 18R; Likang Biological Medical Technology Holding Co., Ltd. Shanghai, China) at 12,000 rpm and 4°C for 20 min, and 400 μl of supernatant was collected. A total of 60 μl of ribose (0.2 mg/ml) was added to each sample as an internal standard. The samples were oscillated in vortex oscillators for 10 s. Next, the samples were dried in a FreeZone Plus 12 vacuum freeze dryer (Beijing Zhongkekeer Instrument Co., Ltd., Beijing, China), and 80 μl of a methoxylamine–pyridine solution (15 mg/ml) was added and oscillated for 30 s. The samples were reacted at 37°C for 90 min in a temperature-controlled electric thermostatic drying oven (DH-101-2BY; Tianjin Zhonghuan Electric Furnace Co., Ltd., Tianjin, China), and 80 μl of BSTFA reagent (with 1% TMCS) was then added quickly. Finally, the samples were incubated in an incubator at 70°C for 60 min. After sample pretreatment, a GC-MS analyzer (GMS-QP2010; Shimadzu Corporation, Kyoto, Japan) was used to detect the samples [8].

Results & Discussion

The main extraction methods of phloem sap include K₂-EDTA exudation and the use of an aphid kiss needle, incisions, centrifugation, etc. [9–11]. The K₂-EDTA exudation method is suitable for most plants and can be used to collect a large amount of phloem sap. The drawback is that the phloem secretions obtained by the K₂-EDTA method may be contaminated by substances from the xylem and other tissues [12]. Aphid kiss needles use insect mouthparts as needles to collect phloem sap. However, sap from the aphid mouthparts contains insect salivary proteins and other components that may affect the chemical composition of the collected phloem sap [13]. The incision method involves cutting the stem of a plant with a blade and collecting the sap exudate from the cross section of the stem. The sap collected by this method consists of not only phloem sap but also a large amount of sap from the xylem and other cells. This method does not accurately locate the position of the phloem sieve tube, so it is difficult to obtain pure phloem sap. The centrifugation method can precisely estimate the composition of phloem sap, but the cell components pollute the phloem sap during centrifugation. None of these methods can extract phloem sap quickly and precisely.

When the syringe and pipette were used to extract phloem sap directly, most of the sap remained in the pipette head and syringe needle, the sap quickly solidified and blocked the extraction equipment, affecting the ability to reuse it. The device designed in this study rapidly extracts sap into a centrifuge tube by air pressure and capillary action before the sap has a chance to solidify, thus allowing the capillary glass tube to be reused [14]. Sugars, the major constituents of phloem sap, were detected by HPLC, whereas other substances were detected by GC-MC.

Sugars detected by HPLC

The sap sampled using the capillary–air pressure and EDTA methods was detected by HPLC. As shown in Figure 6, the concentrations of all sugars sampled using the capillary–air pressure method were higher than those observed using the EDTA method, and the concentrations of sucrose and sorbitol were much higher than other sugars.

Other substances detected by GC-MS

GC-MS was used for qualitative and quantitative analysis of the samples. The molecular and structural formulas of the metabolites corresponding to each peak were obtained using GCMS Real Time Analysis software combined with National Institute of Standards and Technology database information retrieval. The specific name of each substance was determined using the ChemicalBook website (www.chemicalbook.com/).

In the process of sampling phloem sap via the EDTA extraction method, the petiole was placed in a Petri dish containing 10 ml of distilled water for extraction, resulting in diluted phloem sap. Therefore, a partial deviation was used in the determination of phloem sap concentration and substance type extracted using the EDTA method. Sugars, the major constituents of phloem sap, were detected by HPLC, whereas other metabolites were detected by GC-MC. Only substances detected by GC-MS are shown in this article.

Orthogonal partial least squares discriminant analysis

Data were analyzed using SIMCA-P 14.1 software (Umetrics, Umea, Sweden) through orthogonal partial least squares discriminant analysis. Orthogonal partial least squares discriminant analysis was performed on the data detected by the capillary–air pressure and EDTA extraction methods (Figure 7). The tolerance ellipse was constructed using SIMCA-P software based on Hotelling's T². The six observed values of the capillary–air pressure method are shown in Figure 7A, and the six observed values of the EDTA extraction method are shown in Figure 7B. There were no outliers, which means that the data were stable.

Peak value comparison

The initial numbers of peaks obtained by the capillary–air pressure and EDTA extraction methods were 314–334 and 304–327, respectively. The sample data detected by GC-MS were processed to obtain the peak graph with a peak height of more than 5,000,000. As shown in Figure 8A, the number of peaks obtained by the capillary–air pressure method was between 84 and 97, and as shown in Figure 8B, the number of peaks obtained by the EDTA extraction method was between 42 and 49. Figure 8A displays most of the peaks shown in Figure 8B.

As shown in Figure 8, after the samples were treated in the same manner, different chromatograms were obtained using the two different methods. The number of peaks shows that the types of substances extracted using the capillary–air pressure method were significantly more abundant than those extracted using the EDTA method.

Metabolite comparison

The phloem sap of plants contains large amounts of proteins, amino acids and organic and inorganic ions. The inorganic ions in the samples could not be directly detected by GC-MS, and the proteins were removed in the sample pretreatment process. However, amino acids and organic acids are common and easy to detect, so they were used to compare the reliability of the two sampling methods. A total of 46 substances, including organic acids (e.g., oxalic acid, citric acid and malic acid) and amino acids (e.g., glutamic acid, serine and succinic acid), were detected in phloem sap sampled using the capillary–air pressure method. By contrast, 29 substances, including organic acids (e.g., malic acid and oxalic acid), were detected in phloem sap sampled using the EDTA extraction method; however, amino acids were not detected. Details of the detected substances are shown in Table 1.

Table 1 shows that the amount of substances in phloem sap extracted using the capillary–air pressure method was twice that observed in sap extracted using the EDTA method. Therefore, the capillary–air pressure method can be used to extract plant sap for the detection of different kinds of substances.

Contrast of sap substances between the two methods

The inclusion relationships of the samples were extracted by different extraction methods and analyzed by Origin software (Version 2019, Origin Lab, MA, USA). As shown in Figure 9, 46 substances were detected in phloem sap sampled using the capillary–air pressure method and 29 substances were detected in phloem sap sampled using the EDTA extraction method. Of the total number of detected substances, 23 were detected using both methods. Of the substances detected in phloem sap sampled using the capillary–air pressure method, 23 were not detected by the EDTA extraction method. By contrast, only six substances in the phloem sap samples extracted using EDTA were not detected by the capillary–air pressure method. For the samples extracted by these two methods, the detected substances in the samples extracted by capillary-air pressure method include not only most of the detected substances extracted by EDTA, but also the undetectable substances extracted by EDTA.

Substance concentration contrast

Sample concentrations were analyzed using Excel software (Microsoft Corporation, WA, USA). Because of the large difference in magnitude between the two sets of data, the logarithm of the two sets was taken. The sample concentrations of substances obtained using both sampling methods are shown in Figure 10. Ribitol was used as an internal standard, so the concentrations of this substance were the same. The concentrations of substances detected using the capillary–air pressure method were higher than those detected using the EDTA extraction method. The concentrations of the substances obtained using the two methods exhibited similar tendencies.

The same plant was sampled using different methods, and the concentrations of the sample substances were quite different. Because the samples were diluted during sampling with the EDTA method, the concentrations of substances were higher with the capillary–air pressure method than with the EDTA method (Figure 10).

The aforementioned results showed that a greater number of substances were extracted using the capillary–air pressure method compared with the EDTA extraction method, and the concentrations were higher; therefore, the capillary–air pressure method is suitable for extracting phloem sap. In addition, the device designed based on the principle of capillary–air pressure is portable, produces rapid results and is simple to operate. Overall, the method and device can be applied to the rapid extraction of phloem sap.

Future perspective

In future work, the sampling device will be optimized for the extraction of phloem sap from various plant species. The sampling device is slated for real-time plant sap detects, and a portable real-time detection device will be built to obtain more plant information.