Phenolic compound abundance in Pak choi leaves is controlled by salinity and dependent on pH of the leaf apoplast

Abstract Onset of salinity induces the pH of the leaf apoplast of Pak choi transiently to increase over a period of 2 to 3 hr. This pH event causes protein abundances in leaves to increase. Among them are enzymes that are key for the phenylpropanoid pathway. To answer the questions whether this short‐term salt stress also influences contents of the underlying phenylpropanoids and for clarifying as to whether the apoplastic pH transient plays a role for such a putative effect, Pak choi plants were treated with 37.5 mM CaCl2 against a non‐stressed control. A third experimental group, where the leaf apoplast of plants treated with 37.5 mM CaCl2, was clamped in the acidic range by means of infiltration of 5 mM citric acid/sodium citrate (pH 3.6), enabled validation of pH‐dependent effects. Microscopy‐based live cell imaging was used to quantify leaf apoplastic pH in planta. Phenolics were quantified shortly after the formation of the leaf apoplastic pH transient by means of HPLC‐DAD‐ESI‐MS. Results showed that different phenolic compounds were modulated at 150 and 200 min after the onset of chloride salinity. A pH‐independent reduction in phenolic acid abundance as well as an accumulation of phenolic acid:malate conjugates was quantified after 200 min of salt stress. However, at 150 min after the onset of salt stress, flavonoids were significantly reduced by salinity in a pH‐dependent manner. These results provided a strong indication that the pH of the apoplast is a relevant component for the short‐term metabolic response to chloride salinity.


| INTRODUC TI ON
Salt stress is a common abiotic stress that is recognized for its multifaceted negative impact on plant growth. With more than 800 million hectares of salt-affected land worldwide (Munns & Tester, 2008) and in respect of the ever growing demand for food and fodder crops, plant scientist are encouraged to develop a set of means to warrant food security.
Salinity affects plants in two ways. First, it reduces the osmotic potential of the soil solution, decreasing water and nutrient uptake by plants. This osmotic stress component acts fast (hours to days) and reduces the rates by which shoots, leaves and to a lesser degree roots expand (Bartels & Sunkar, 2005;Feng et al., 2018). Adaptions either comprises increased net rate of osmolyte deposition (e.g. hexoses, K + ) or reduced cell expansion (Sharp et al., 1990). In other words, the plant struggles to take up water and strives to lower the cellular osmotic potential in order to facilitate water uptake (Zörb et al., 2019). In the longer term (days to month), excessive amounts of salt ions accumulate in the tissue. This second so-called ion toxic phase is characterized by a dysfunctional photosynthetic machinery (Bose et al., 2017) that gives rise to the formation and accumulation of reactive oxygen species. These radicals damage plasma membranes, proteins or DNA by oxidation (Mittler, 2017). Excessive concentration of the salt ions in the cytosol is also suggested to inhibit activities of enzymes (Flowers et al., 2010). As reviewed by Munns and Tester (2008), ion-specific salt-tolerance is often acquired by at least one of the two strategies: (i) The exclusion of ions by roots, which avoids accumulation of ions in the shoot and (ii) the ability of leaf tissue to tolerate high ion concentrations by ion sequestration into the vacuole.
Overall, much is known about these mid to long-term stress responses, that is, about the osmotic and the ionic phase, whereas information about the fast response that ensue minutes to hours after the onset of salt stress is scanty. There are a few studies on fast salinity-induced changes of gene expression and proteome patterns, suggesting for rice that this early phase is indeed important for adaptation (time frame: 15 min to 6 hr; (Kawasaki et al., 2001;Roshandel & Flowers, 2009;Zhang et al., 2009)). Likewise, it was reported that changes of the gene and miRNA (Hernandez et al., 2020;Popova et al., 2008;Ueda et al., 2004) expression profile, as well as of the metabolome (Kim et al., 2007;Wang et al., 2009) coincide with the earliest phase of salt stress.
Another short-term effect comprises changes in the pH of the leaf apoplast. Recently, we reported about changes of the apoplastic pH (pH apo ) in leaves of Vicia faba that were formed at 20 min after plants were challenged by salinity at the root level. These pH changes were induced by the chloride-component of NaCl salinity being able to modulate the (i) distribution (Geilfus et al., 2015) and (ii) gene expression (Geilfus et al., 2020) of the phytohormone abscisic acid (ABA), causing stomata to close. This transient alkalinization of the apoplast also induced changes of the leaf proteome that were detectable after 2 to 3 hr (Geilfus et al., 2017). Among the saltresponsive proteins were enzymes of the branched phenylpropanoid pathway, namely phenylalanine ammonia lyases (PALs) and the cinnamyl alcohol dehydrogenase (CAD). Both are pivotal for the synthesis of polyphenols (e.g. flavonoids) (Winkel-Shirley, 2002) and monolignols (Halpin et al., 1994) and are likely to be key enzymes for stress adaptations. It is not known whether these compounds are affected quantitatively during the onset of chloride salinity in a pH apo -dependent mode.
This study was conducted in order to investigate whether a short-term chloride salinity treatment modulates abundances of compounds that are synthesized by the phenylpropanoid pathway.
Second, it aimed at clarifying the role of the leaf pH apo for such putative responses. We studied this response in Pak choi [Brassica campestris L. ssp. chinensis (L.) Makino] because it is an important leafy vegetable for human nutrition that is rich in phenylpropanoids (Harbaum et al., 2007). By means of HPLC-analyses of secondary metabolites, together with ratiometric real-time in-planta measurements of pH in intact plants, the present study demonstrates that salinity reduced flavonoid contents in a pH apo -dependent manner, whereas changes of phenolic acids were found to be pH apo -independent. CaCl 2 to the roots. However, before the stress treatment was initiated, the leaf pH apo of the fourth oldest leaf was clamped in the acid range via infiltration of 5 mM citric acid/sodium citrate (pH 3.6).

| Plant cultivation and experimental design
Plants from the third group (n = 5) represent the non-stressed controls (neither CaCl 2 to the roots nor buffer infiltration). The fourth oldest leaf of plants of group 1 and 3 was infiltrated with water as a control to check the effects of the infiltration procedure. At 150 and 200 min after the addition of 37.5 mM CaCl 2 to the roots, leaves were harvested, immediately shock frosted and freeze-dried to determine the contents of flavonoids and phenolic acids. The leaf apoplastic pH was measured continuously every 10 min over the entire 200 min of the experiment using live cell imaging. Leaf chloride content was measured every 50 min. While pH apo measurement was conducted non-invasively via optical methods, measurements of leaf chloride content and metabolites were performed destructively, requiring a separate batch of plants for each time point. In the present work, all analyses were performed on the fourth oldest leaf (n = 5 biological replicates).

| Inverse microscopy imaging
The leaf pH apo was quantified in planta via apoplastic H + -live-imaging using a microscopy-based ratiometric approach. In brief, the dextranated (10 kDa) pH-sensitive dye Oregon Green 488 (Invitorgen GmbH, Darmstadt, Germany; dissolved in deionized water) was used as an apoplastic pH-sensor. For this, it was infiltrated through the open stomata into the apoplast as described by Geilfus and Mühling (2011). Dye signals were detected with a fluorescence microscope (DMI6000B; Leica Microsystems, Wetzlar, Germany) using a dry objective (HCX PL FLUOTAR L, Leica Microsystems, Wetzlar, Germany) and a DFC-camera. Dye was exposed for 25 mS at the excitation wavelengths of 440/20 (pH-insensitive) and 495/10 nm (pHsensitive). Light emission at both channels was collect at 535/25 nm.
As a measure of pH, the fluorescence ratio F 495 /F 440 was calculated.
Ratio values were converted into pH values via an in vivo calibration (Geilfus et al., 2014).

| Analysis of leaf Cl − contents
Leaf Cl − contents were quantified in 15 mg dried leaf samples. For extraction, milled leaf samples were boiled for 5 min in 1.6 ml deionized water. After being cooled down on ice, the samples were centrifuged. The supernatant was collected and proteins were removed by chloroform precipitation. Hydrophobic compounds were removed by a passage through a C18-E column (Strata, Phenomenex, Torrance, CA, USA). Purified samples were ready for the measurement of chloride concentration using ion chromatography (ICS 5000,Thermo-Dionex, Sunnyvale, CA, USA) as described elsewhere (Geilfus et al., 2017).

| Determination of flavonoids and phenolic acids
To analyze the profile of selected phenolic acids and flavonoids in the Pak choi leaves, 20 mg lyophilized powder was used for the extraction. Based on a method described by Förster et al. (2015), the leaf powder was extracted in 300 µl of 70% methanol (pH 4, acetic acid) for 15 min in ice water using sonification (Bandelin Sonorex). The pellet was re-extracted twice with 300 µl of the extraction solvent for 10 min. After each extraction step, the samples were centrifuged for 5 min at 16,000 g (Thermo Scientific, Heraeus Megafuge X1R Centrifuge) at 4°C and the supernatants were combined. Supernatants were concentrated (vacuum concentrator, Thermo Scientific Savant SPD111V Concentrator, vacuum pump: Vacuubrand PC 3001 series, CVC3000) to near dryness, dissolved in 50% methanol, and filled up to 1 ml. The samples were shortly vortexed and centrifuged for 10 min at 16,000 g, filtered (Costar ® SpinX tubes), and transferred to HPLC vials. Phenolic acids and flavonoids in extracts were qualitatively and quantitatively analyzed by HPLC (Ultimate 3,000, Thermo Scientific). A volume of 10 µl extract was injected and separated using a 150 x 2.1 mm C16 column (AcclaimPA, 3 μm, Thermo Scientific). Two solvents were used for analysis: solvent A: H 2 O (0.5% formic acid), B: 40% acetonitrile. For separation, the following gradient program was used: 0-1 min: 0.5% B, 1-10 min: 0.5%-40% B, 10-12 min: 40% B, 12-

| Data evaluation
Statistical analyses were conducted in R (R Core Team, 2014) and figures were produced using the package ggplot2 (Wickham, 2009).
Data are presented as mean ± standard error of mean (SEM). For each secondary plant metabolite and sampling time point the effect of treatment was determined by a one-way ANOVA (α = 0.05). Pvalues were adjusted according to the Bonferroni correction method for multiple comparisons. When effects were significant, Tukey's HSD test was performed to discriminate between treatment groups.

| Ratiometric in planta monitoring of leaf apoplastic pH
The ratiometric pH quantification revealed a transient alkalinization of the leaf apoplast when intact Pak choi plants were treated with 37.5 mM CaCl 2 at the root level. Starting from a pH apo of 4.3, a rise occurred for 60 min, resulting in a maximum pH of 6.1. After 10 min of stagnation, the pH steadily decreased to 3.9 and ultimately stabilized at the initial pH of 4.3 (Figure 1). This transient alkalinization could be inhibited by the infiltration of a pH-buffer (5 mM citric acid/sodium citrate) into the leaf apoplast prior to exposing roots to 37.5 mM CaCl 2 . However, this pH-buffering lowered the pH apo to 3.6. The pH apo in the non-stressed control groups was stable at 4.4 over the entire experiment (Figure 1).

| Leaf chloride concentration
Under control conditions, Pak choi leaves contained 2 mg Cl − g −1 dry weight (DW) (Figure 1; red kinetics). The addition of 37.5 mM CaCl 2 to the roots induced a fast accumulation of Cl − . Already after 50 min, measurements revealed a significant increase to 6.2 mg Cl − g −1 leaf DW, reaching a content of 8.3 mg Cl − g −1 leaf DW after 200 min.
The pH-buffering of the leaf apoplast provoked a less steep increase in Cl − compared to the non-pH apo -buffered experimental group that was stressed with CaCl 2 , resulting in lower content of 6.9 mg Cl − g −1 leaf DW after 200 min.

| Quantification of flavonoid and phenolic acid derivatives
HPLC analysis identified three flavonoids (FLs) and eight phenolic acid (PA) derivatives in leaves of Pak choi with contents ranging from 0.004 to 5.781 µmol/g DW (see Table 1 Of the eight PAs, four derivatives were identified as malate conjugates. Summation of the single metabolites of the free total phenolic acids (total PA) as well as the total phenolic acid:malate conjugates (total PAmC) revealed similar trends with respect to the CaCl 2 -effect on FL contents, however, not being significant (Table 1)

| D ISCUSS I ON
According to Lager et al. (2010), changes in the external pH rapidly alter expression of many genes as shown for roots of Arabidopsis thaliana. In agreement with these results, we have found that In the presented study, real-time in-planta pH monitoring was used to demonstrate that the leaf pH apo of Pak choi can also be modulated by means of chlorine salinity (Figure 1; black kinetic). Previous studies with chloride-accompanying counterions revealed that the transient pH-increase is attributable to the anion (i.e. Cl − ) and not to the accompanying cation or the osmotic compound of the stress treatment (Geilfus & Mühling, 2013). The transient increase in pH apo that followed the CaCl 2 -treatment could be inhibited by clamping of the apoplast in the acidic range. Both groups (37.5 mM CaCl 2 vs. 37.5 mM CaCl 2 + pH buffer) accumulated Cl − contents which were far above the requirements for chlorine as a micronutrient (Geilfus, 2018), indicating the beginning of excessive exposure of the tissue to Cl − .

Results
HFM is a PA that is argued to be an antifungal agent (Quentin et al., 2014) and was found to be increasingly synthesized in three different Brassica rapa cultivars, upon fungal infection (Abdel-Farid et al., 2009). Here, we found an HFM accumulation provoked by abiotic stress. Further insights on the physiological and biochemical role of HFM as well as the PAmCs in general, is scanty.
Therefore, implications for functional traits linked to an accumulation of PAmC are hardly possible.
The contents of IRG, KCDG, and KHDG were not influenced by salinity. Similar to the marked dynamic of total FLs, IRG, a major constituent of the phenolics in Brassica (Romani et al., 2006), decreased by 58%, 150 min after the addition of 37.5 mM CaCl 2 to the roots.
Yet, due to great variances in the control and fixed-pH salt stressed treatment group, no significant effect could be deduced.

| SUMMARY
The aim of this study was to evaluate the role of pH of the leaf apo- and FLs, which were mediated by pH apo . This is the first evidence that salinity-induced changes of the leaf apoplastic pH are functional with respect to the modulation of metabolite abundances.
Additionally, PAs that are conjugated to malate were affected in a reciprocal way by salt when compared to non-malate conjugates.
This implies that these PAmCs comprise a salt-responsive subgroup of phenolics, an observation that, to our best knowledge, had never been reported before.

ACK N OWLED G M ENTS
This work was supported by a BMBF research grant (CUBES Circle 031B0733A) and by a DFG research grant (GE 3111/1-1), both organisations are gratefully acknowledged.

CO N FLI C T O F I NTE R E S T
Authors have no conflict of interest to declare.