A new device for online nanoscale sampling and capillary electrophoresis analysis of plant sap composition

In this work, an online sampling of plant xylem sap combined with an efficient (CE)‐based method was developed and applied to study the kinetics of changes in the sap composition and to assess plant fitness under stress conditions comprehensively. A laboratory‐built CE device was developed to provide online sampling and CE analysis of various ionogenic species in the sap during plant stress response. The rapid online sampling and short CE analysis time allow for real‐time monitoring of changes in sap constituents in the living plant during the stress response. The developed device was successfully used to analyze chloride, nitrate, and sulfate ions in the plant xylem during the salt stress or stress caused by nitrate deficiency within short time scales.

plants are exposed include external factors such as light quality and quantity, drought, ambient temperatures, and environmental pollutants.Drought and constantly rising temperatures are the factors that most limit plant growth.To overcome these limitations, it is necessary to get insight into the complex response of plants to stress stimuli involving the communication structure of environmental and intrinsic signaling pathways [2,3].
A valuable way to gain insight into plants' physiological and nutritional status is to analyze the composition of their conductive vascular tissues, that is, phloem and xylem.The xylem ensures the transport of water, mineral nutrients, metabolites, and signaling molecules from the root to the transpiring parts of the shoot, especially to the photosynthetically active leaves [4].On the other hand, the phloem is responsible for transporting assimilates from the sites of production, for example, photosynthetically active parts, to the growing areas of the shoot and root [5].Previously, an extensive overview of recently published methods for the analysis of phloem or xylem sap components using capillary electrophoresis (CE) methods was given, discussing them according to the main groups of analytes and briefly presenting the contribution of the obtained results to the current understanding of plant physiology and nutrition processes [6].
The complexity of the matrix of plant saps and the variability of their composition make the analysis even more challenging, as the amount of the sap sample obtained is often minimal in most plant species, only nanoliters or microliters [7].In contrast to methods that provide us with average concentrations of all analytes determined in the homogenized tissue and often require preselection of the target group of analytes [8,9], low-invasive and -sampling methods could be employed in combination with CE methods.Besides the fact that samples analyzed by CE could be introduced into the separation capillary with none or minimal sample pretreatment, the main advantages are high separation efficiency and negligible sample and reagent consumption.CE can also be automated and easily coupled with various detection methods [10].This approach allows us to examine plants even at the early stages of development and also with minimum damage.Moreover, the composition of xylem sap can change rapidly and significantly affect leaf physiology, as was previously demonstrated after salt treatment [11].Rapid sampling and analysis of sap, hence, provide a valuable tool for uncovering details of the physiological response.Finally, comprehensive information can be obtained on the current state of the plant's fitness during growth and/or under stress conditions.
In this work, we describe practical testing of a laboratory-built CE device that was developed and fabricated to provide a low-invasive nanoscale sampling of plant sap, followed by CE analysis of various ionogenic species in plant xylem during the early stages of the stress response in short time scales.

Description of CE instrumentation
The instrumentation used for electrophoretic separations comprised a laboratory-built CE device with two BGE reservoirs connected to an HV power supply (Villa Labeco, s.r.o.) via two inserted platinum wire electrodes with a diameter of 0.3 mm and a length of 2 cm (SAFINA, a.s.), and banana-type connectors (GM electronic, spol.s r.o.).The analytes were detected using a conductivity detector (C4D, TraceDec, Innovative Sensor Technologies GmbH).Data were collected and evaluated using an external 24-bit A/D converter U-PAD2 (DataApex) and Clarity Lite software (version 2.8.01.584,DataApex).A commercial CE system (7100 CE System, Agilent) with C4D was also used for sample analyses.The C4D cell was placed in the capillary cassette, and the detector module was connected to the Agilent CE system via the analog in port to collect data.The CE separations were performed in a Polymicro fused silica capillary (50 µm ID, 375 µm OD, Molex, Lisle).The total length of the capillary was 50 cm, and the effective length was 35 cm for both the laboratory-built CE device and the commercial CE system.

CZE analysis of inorganic anions in plant xylem
All xylem samples were analyzed as collected without any dilution/purification.BGE consisted of 100 mM MES/His, pH 6.1, and 30 µM CTAB.The separation capillary was conditioned at the beginning of each day with a solution of 1 M sodium hydroxide for 5 min, deionized water for 5 min, and BGE for 10 min.Between each analysis, the separation capillary was washed by BGE for 2 min.For every six analyses, the separation capillary was flushed with 1 M sodium hydroxide solution for 1 min, deionized water for 3 min, and BGE for 5 min.The samples were injected hydrodynamically (40 s siphoning, a 6 cm height difference for the laboratory-built CE device, and 5 s at 50 mbar for the commercial CE system).The separations were performed at −30 kV.

Plant material and instrumentation for xylem sap collection
Seeds of Brassica napus were germinated on moist cellulose tissue and transferred to hydroponic cultivation after 2 weeks.Plants were grown in containers with a modified Hoagland solution containing 2 mM nitrate as the sole source of nitrogen (N) (see Table S1 for details of nutrient solution composition).The solution was continuously mixed and aerated.Cultivation was carried out in a climate chamber with a day/night temperature of 22/18 • C, a photosynthetic photon flux density of 500 µmol/m 2 /s, a 14-h photoperiod, and 60% relative air humidity.

Determination of the correct sap flow rate and its effect on xylem sap composition
The root pressurization technique was used for the extraction of the xylem sap samples, and its assumption was confirmed for Brassica plants [12].The plant was placed in a beaker filled with a nutrient solution in a climate chamber for 1-2 h.The plant with the beaker was weighed at the beginning and end of the period.The decrease in the weight over time was a proxy of the rate of water transport in the plant supporting transpiration from the leaves.A young fully developed leaf was then cut from the plant, and the leaf water potential (WP L ) was determined using a pressure chamber (Model 3005 Plant water status console, Soil moisture Equipment Corp.).The plant was transferred to a new beaker filled with a fresh aerated nutrient solution and placed in the pressure chamber.The shoot of the plant was cut off below the oldest leaf and the chamber was closed with the rest of the stem above the root system sealed in the chamber lid.The pressure was continuously increased.When the first drop of the sap appeared on the cut surface of the stump, the pressure increase was stopped, and the sap was collected by a pipette for 1-3 min.The sap flux rate at the appropriate pressure level was calculated from the weight of collected sap in the vial and the exact length of the sampling period.The described procedure was repeated at different pressure levels (4-6 per plant) with an estimated value of WP L in the middle of the range.The calculated sap flow rates at a pressure equal to WP L were then correlated with the sap transport rates of intact plants.In addition, aliquots collected at different pressure values were analyzed with the commercial CE system to show the effect of sap flux rate on the concentration of ions in the xylem of Brassica plants.

2.4.2
Xylem sap sampling during the plant stress response The first type of experiment was focused on the response of plants to N deficiency under controlled xylem extraction conditions.Nine plants cultivated in full nutrient solution were transferred at the beginning (time "0 min") to an N-free solution with a similar ionic composition, where only nitrate was replaced by sulfate and chloride (Table S1).Twelve plants transferred to the same fresh nutrient solution were used as control treatments to account for the effect of mechanical disturbance to the plants and daily changes in uptake rates.Sampling was carried out at 0, 2, 4, and 6 h after the start of the experiment.After the determination of plant WP L with one cut leaf, the root system was detached and pressurized in the corresponding solution in the pressure chamber to a pressure equal to WP L .The cut surface of the stump was washed with distilled water, and the first drop of sap was discarded.Aliquots of sap (30-100 µL) were then collected and analyzed by the commercial CE system.
In the second type of experiment, the laboratorybuilt CE device and online sap sampling method were used to monitor the response of intact plants to Ndeficiency/increased NaCl concentration in the nutrient solution.Three plants were analyzed within each treatment.This experiment was performed in a custom-built pressure chamber that accommodated an intact plant with roots in a beaker filled with a full nutrient solution.The stem of the plant was sealed in the lid with an elastic silicon impression material.After the plant was placed in the chamber, one leaf was cut off and used to determine the WP L value.The cut petiole of the plant was then used for sampling the expressed sap.The first drop of xylem sap was discarded, and the cut surface of the petiole was washed with distilled water to remove any contaminants from the cut cells.A silicone tubing was placed on the petiole to create a reservoir and prevent sap evaporation.The pressure constantly applied to the plant root system was close to the numeric value of WP L and allowed a droplet of xylem sap to appear at the end of the cut petiole.The pressure applied during sampling was slightly higher (0.1-0.2 MPa) than WP L to allow the collection of approximately 30-50 µL of the sap.After sampling, the pressure was decreased again to stop the flow of sap from the petiole.The sampling procedure was repeated every 15-20 min.Aliquots were analyzed by the laboratory-built CE device in situ, and the remaining volume of each sample was later analyzed by the commercial CE system as reference.Samples were stored at 4 • C or frozen if not analyzed within 24 h.

Design and fabrication of the laboratory-built CE device
The laboratory-built CE device (Figure 1A) was developed to provide both online sampling and CE analysis of various ionogenic species in the plant sap.The 3D model of the designed device was created using the Autodesk Inventor software (Autodesk).The device consists of a base plate with two buffer reservoirs and a mounted manipulator for separation capillary.The buffer reservoirs and additive capillary tubing were fabricated from PEI 1000 polymer (Tribon s.r.o.).The base plate fabricated from material PE-500 (Tribon) is attached to the top of a UV detector surrounding its detection cell (note: UV detector is a part of the laboratory-built CE device but is not used within experiments presented in this article).A C4D cell is secured within the base plate with a purpose-made screw.The screws needed for assembling the CE device were made of polyamide PA-66 material (Plastové součástky s.r.o.), and all purpose-made screws for fixing the separation capillary were fabricated from Ketron PEEK-1000 material (Tribon).The sampling needle is represented by the tip of a separation capillary having its inner diameter.The sampling needle is provided with a manipulator consisting of a fixed and moving arm.The moving arm has its first end connected to the fixed arm through a first rotating joint.The second end of the moving arm is provided with a second rotating joint equipped with a through-hole and a purpose-made two-piece screw for fixing the sampling needle in the desired position.The moving arm ensures moving the sampling needle between the sampling position, that is, sampling of sap (Figure 1B), and the resting position (Figure 1D), that is, the end of the sampling needle is in the inlet buffer reservoir and taking up a buffer.The separation capillary leads into the additive capillary tubing with two outlets and is fixed with a purpose-made screw.The first outlet is equipped with silicone tubing, a two-way valve, and a pump represented by a plastic syringe.When the flow continues toward the pump, the two-way valve is open.In combination with a negative pressure created by the pump at the outlet side of the separation capillary, the capillary is flushed by BGE from the inlet side, and the liquid is sent to the waste.The second outlet of the capillary tubing is provided with the outlet buffer reservoir with a silicone valve.When negative pressure is applied to flush the capillary, the silicone valve closes the second outlet.On the other hand, the two-way valve is closed, the silicone valve is floating freely in the BGE within the second outlet, the whole system is filled with a BGE, and the separation may start.Both buffer reservoirs are equipped with platinum electrodes connected to a high-voltage power supply through banana-type connectors.

CE analysis of small inorganic ions
Small inorganic ions were analyzed in plant xylem by CE with conductivity detection.BGE consisted of an MES/His buffer, pH 6.1, and an electroosmotic flow-modifier CTAB was selected [13,14].The composition of BGE was optimized to fulfill the requirement of direct analysis of the plant xylem, which means an injection of the sample without adjustments from the source origin and to ensure sufficient peak resolution.The optimized BGE consisted of 100 mM MES/His, pH 6.1, with the addition of 30 µM CTAB.Despite the high ionic strength of BGE, 30 kV could be used for analyses due to the low BGE conductivity (28 µA and 0.4 W/m), and all the ions monitored were detected within a few minutes.The typical electropherograms obtained by the commercial Agilent CE system and/or laboratory-built device are shown in Figure S2.
Limit of detection (LOD) and limit of quantitation (LOQ) of CE analysis using the developed laboratory-built CE device were determined from the calibration curves performed in triplicates with the good linearity in the range of 7-130 µM (R 2 > 0.95).For chloride, nitrate, and sulfate ions, LODs of 0.015, 0.018, and 0.012 mM (S/N = 3) and LOQs of 0.064, 0.076, and 0.048 mM (S/N = 10) were determined.

Application of the laboratory-built CE device in biological experiments
We aimed to demonstrate the feasibility of using the laboratory-built CE device for monitoring the xylem composition in real time at time scales relevant to the dynamics of plant response under stress.Two different types of abiotic stress responses of Brassica plants were monitored, namely, responses to N deficiency stress and sodium chloride salt stress.Important issues related to sample collection were addressed before the practical use of the instrument.The technique of sap sampling can significantly affect the composition of xylem sap [15].Particularly, the flow rate of sap has a significant impact on the concentration of sap constituents.Therefore, to obtain physiologically relevant information, sampling should be performed at a flow rate that corresponds to the transpiration rate (TR) of the plant under the cultivation conditions.Before biological experiments, we successfully verified the previously published sap sampling method for use with Brassica plants [12].Their results showed that the pressure numerically equal to WP L promotes the flux of sap that corresponds to the rate of water transport in plants at the corresponding steady-state TRs.We found a highly significant linear correlation between the whole plant TR and the sap flux rate from the detached root system pressurized by pressure numerically equal to WP L (Figure S1).This indicates that the WP L of Brassica plants in hydroponic culture can serve as a good predictor of the driving force necessary to promote the actual transpiration flow of the plant under study.Therefore, we used the pressure derived from WP L for sap sampling to ensure that sap composition precisely reflected changes in the plant during the stress treatments.As part of the method development, we also investigated possible errors resulting from sap sampling at rates higher or lower than the actual TR of the plant.Aliquots of sap collected at a range of applied pressures were analyzed on the commercial CE system.The results confirmed that ion concentration decreased with increasing sampling pressure (Table S2).Because radial transport of water and ions in the root are independent processes, at higher sampling pressures, water is displaced from the surrounding solution and root cells into the conductive tissues and dilutes the sap.Conversely, when the sap flow rate is slower than the TR (lower pressures), the xylem sample is more concentrated.Our results show that this kind of error is greater when the sap flux is lower than the TR (up to 120% deviation from true concentration) than when the sap flux is higher (up to 30% deviation; Table S2).This indicates that a small increase of pressure above WP L during sampling caused only a negligible deviation of ionic concentrations in sap from steady-state conditions.

3.3.1
Monitoring of anions in the xylem during nitrogen deficiency stress Real-time detection of changes in the composition of transported solutes in plants represents a major methodological challenge in plant science.Small sample volumes, large differences in the concentrations of examined ions, or variable response time scales are just a few examples of complicating factors.
The external availability of nitrate as an important nutrient and signaling molecule is promptly reflected in uptake and transport in the plant [16].There has been discussion on how rapidly the changes in availability may be apparent in the export of nitrate from root to shoot via xylem.The nitrate uptake by root cells and xylem loading are independently regulated processes, and the nitrate storage in root cells can buffer the imbalance between rates of uptake and xylem transport [17].In the first type of experiment, we attempted to detect changes in xylem sap composition in plants transferred to media without N on a short time scale of a few hours on 12 controls and 9 treated plants.The nitrate level dropped significantly (p < 0.05) as early as 2 h in the N-free medium.Simultaneously, chloride and sulfate levels increased significantly (Figure S3), suggesting that the stimulated export of these anions to the xylem compensated for reduced nitrate transport to maintain the balance between cations and anions [5].
The second type of experiment aimed at online monitoring the response of three intact plants to N-deficiency.Based on the results of the previous experiment, the changes in the sap composition were monitored more in detail for 2 h after the start of the treatment when a significant drop in ion concentration was observed.Due to the higher variability of the manual injection using the laboratory-built CE device, which has to be considered, we used the ratio of chloride to nitrate peak areas to detect changes in xylem ion concentrations over time.This approach was compared with the analysis of collected xylem aliquots by the commercial CE system, where individual ions were quantified (based on the calibration curves) and chloride/nitrate ratios were calculated.The results of analyses show approximately a 50% increase in the chloride-to-nitrate ratio, and the data obtained by the commercial CE system reliably confirmed the results from the laboratory-built CE device (Figure 2).The observed change in xylem sap composition following a change in external nutrient availability is rapid, and root cells do not provide much buffering capacity for nitrate transport.Our analytical device proved to reliably detect the fine changes in xylem sap due to the altered composition of the external medium.

Monitoring of anions in the xylem during salt stress
In the following experiments, the response of Brassica plants to salinity induced by an elevated concentration of F I G U R E 3 Anion concentration changes in the xylem sap of three hydroponic Brassica plants during the salt stress.Analyses were performed using the laboratory-built capillary electrophoresis (CE) device (orange circle) and the commercial Agilent CE system (blue square).sodium chloride (50 mM) in a full Hoagland medium was monitored.The plant response after salt application can be very fast, and changes in leaf physiology can be observed in less than 1 h [11].The laboratory-built CE device allowed sampling in 15-min intervals for 1.5 h after salt addition.We were able to easily monitor relative changes in ratios between major anions in this rather tight timeframe (Figure 3).
The increase of NaCl concentration in the nutrient solution to 50 mM affected the osmolality of the nutrient solution and water uptake into the root.In the sampling system where a whole intact plant was used, we balanced the increased osmolality by increasing the pressure in the chamber with the root system.This setting compensated for the lowered water potential of the nutrient solution and allowed for nonrestricted water uptake both before and after salt addition so that the xylem flux was not significantly changed.
The time resolution of sap sampling less than 1 h is important in these kinds of experiments.Our data indicate the greatest changes in xylem composition occurred within 30 min after salt addition (Figure 3), and the increase in the chloride-to-nitrate ratio was approximately 50% of the total increase during the experiment.The reference sap aliquots collected in parallel and analyzed with the commercial CE system showed similar trends in changes in sap composition.Although chloride is an essential micronutrient for plants, in higher concentrations it can be toxic.It causes severe ion imbalance and osmotic stress and interferes with the nitrate uptake and transport in plant [18].We suggest that the impact of elevated external chloride concentration on nitrate uptake and long-distance transport can be reliably detected by our CE device in less than 1 h after salt addition.

CONCLUDING REMARKS
The laboratory-built CE device was designed and manufactured for online sampling and CE analysis.This system was used to analyze the composition of the xylem sap of Brassica plants and to monitor the changes in chloride, nitrate, and sulfate ion concentrations as a response to a stress stimulus.The plant xylem sap was obtained by pressurizing the root system and collected in a reservoir from where it was injected directly into the separation capillary.The developed device proved to be a useful tool for real-time monitoring of changes in sap composition due to stress treatments.Due to the main characteristics such as ease of sampling and short analytical time, the device corresponds well to the required timing of analysis in practical experiments, as demonstrated by monitoring plant response to nitrate deficiency or sodium chloride salt stress.Based on the presented proof of concept experiments, the developed instrumentation proved to be a useful tool for observing changes in sap composition due to the stress treatment in real time.The developed sampling device could find wider applicability in the field of plant physiology, especially in monitoring nutrient delivery to various parts of the plant shoot via xylem.

A C K N O W L E D G M E N T S
The financial support from the European Regional Development Fund-Project "SINGING PLANT" (No. CZ.02.1.01/0.0/0.0/16_026/0008446) and the Czech Academy of Sciences (No. RVO:68081715) is acknowledged.
Open access funding provided by CzechELib.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors have declared no conflict of interest.

D ATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

F I G U R E 1
The laboratory-built capillary electrophoresis (CE) device for online sampling and CE analysis: (A)-a photograph of the instrumentation setup; a-base plate; b-buffer reservoir (b 1 -inlet; b 2 -outlet); c-manipulator; d-separation capillary; e-platinum electrodes + connectors; f-additive capillary tubing; g-high-voltage power supply; h-C4D; i-computer; j-A/D converter; (B)-a photograph of the moving arm with the sampling needle in the injection position; (C)-a detail of the sampling needle during sample injection; (D)-a photograph of the moving arm with the sampling needle in the analysis position.

F I G U R E 2
Anion concentration changes in the xylem sap of three hydroponic Brassica plants during the nitrogen deficiency stress.Analyses were performed using the laboratory-built capillary electrophoresis (CE) device (orange circle) and the commercial Agilent CE system (blue square).