Tissue ionome response to rhizosphere pH and aluminum in tea plants (Camellia sinensis L.), a species adapted to acidic soils

Abstract The growth of tea plants (Camellia sinensis L.) is promoted by the presence of aluminum (Al), a beneficial element under acidic conditions, but the influence of rhizosphere pH on this interaction is not known. To understand the mechanisms underlying the adaptation to acidic rhizosphere conditions, we evaluated ionome profiles and the effect of pH on tea growth in hydroponic culture. The optimum pH for tea growth was around pH 4.2, and growth was inferior under a pH less than 3.8 or higher than 5.0. Under the optimum pH growth and Al accumulation were markedly stimulated by Al treatment. Al content and accumulation in new and mature leaves and new roots (the predominant tissues that accumulate minerals in tea plants) gradually declined with decrease in pH, especially in new roots. Ionome profiles drastically altered Al treatment, but changes were more pronounced in new roots than in new or mature leaves and did not depend on pH. Although the uptake of most cationic minerals in new roots was decreased by Al treatment, cationic mineral contents in new and mature leaves were not decreased by Al. In contrast to other plant species, the content and accumulation of manganese, despite it being a cationic nutrient, were significantly increased by Al treatment. These results indicated that one role of Al as a beneficial element was to maintain the shoot nutrient status by effectively utilizing Al‐limited elements in the roots.

Tea plants grow well in acidic soils because large amounts of Al can accumulate throughout the plant, especially in mature leaves, but stimulates rather than inhibits growth (Ghanati, Morita, & Yokota, 2005;Konishi, Miyamoto, & Taki, 1985;Matsumoto et al., 1976;Morita, Yanagisawa, Takatsu, Maeda, & Hiradate, 2008;Sun et al., 2020). In particular, Al promotes new root growth through maintenance of DNA integrity in root meristematic cells (Sun et al., 2020). Hence, Al is considered to be a beneficial element for tea plants. The growth of tea plants may be stimulated by Al-induced increase in the activities of antioxidant enzymes, resulting in enhanced membrane integrity and delayed lignification and aging (Ghanati et al., 2005). Hajiboland, Bahrami-Rad, Barceló, and Poschenrieder (2013) reported that tea plants showed increased antioxidant defenses and a higher photosynthesis rate mediated by Al. Binding of Al to cell wall-bound phenolic acids would reduce their availability for subsequent enzymatic reactions and might lead to lower lignin content (Hajiboland, Bastani, Bahrami-Rad, & Poschenrieder, 2015). However, the detailed roles of Al as a beneficial element for tea plants remain unknown.
To improve the yield and quality of tea leaves, tea fields, especially in Japan, tend to receive higher rates of nitrogen (N) fertilizer than other vegetable crops, generally as ammonium sulfate and sometimes exceeding 1000 kg N ha −1 year −1 (Akiyama, Yan, & Yagi, 2006;Tokuda & Hayatsu, 2004). Heavy use of ammonium sulfate also causes soil acidification as a result of the accumulation of sulfate ions and nitrification (Tachibana, Yoshikawa, & Ikeda, 1995), sometimes leading to such strongly acidic soil with a pH less than 3.0 (Tokuda & Hayatsu, 2004). It is considered that the optimum soil pH for tea cultivation is around pH 4-5, but many tea fields do not meet this standard. As mentioned above, the soil pH also affects the plant response to Al. To achieve sustainable and stable tea cultivation, it is necessary to establish a balance between changes in the degree of Al activity in response to rhizosphere pH changes and tea growth.
The ionome is defined as the mineral nutrient and trace element composition of an organism, representing the inorganic component of cellular and organismal systems (Salt, Baxter, & Lahner, 2008). Ionomics involves quantitative measurement of the elemental composition of organs or tissues and requires the application of high-throughput elemental analysis technologies using inductively coupled plasma-atom/optical emission spectrometry (ICP-AES/OES), ICP-mass spectrometry (ICP-MS), X-ray fluorescence, and neutron activation analysis, and their integration with bioinformatic analysis (Salt et al., 2008). Ionomics is a useful tool to understand physiological processes because plants first perceive minerals in the rhizosphere, and alteration in any processes that transport inorganic ions from the soil solution to the plant body may affect the plant's ionome . Multivariate ionomic signatures were established to define physiological responses such as iron (Fe) and P homeostasis . Furthermore, dynamic alterations in the ionome have been confirmed in response to environmental factors including temperature (Quadir, Watanabe, Chen, Osaki, & Shinano, 2011), salt stress (Wu et al., 2013), and N status (Chu et al., 2016).
As mentioned, Al stress can affect the cellular homeostasis of various ions (Babourina & Rengel, 2009;Bose et al., 2010aBose et al., , 2010bBose et al., , 2011Bose et al., , 2013Plieth et al., 1999;Rengel & Zhang, 2003). For an Al accumulator species, it is possible that ion homeostasis is optimized to maintain or promote growth while accumulating Al. In the present study, we studied the effects of acidic pH and Al on the growth and tissue ionome dynamics of tea plants in hydroponic culture to determine the optimum rhizosphere pH and investigate the beneficial roles of Al. The results showed that the alteration to ionome profiles in tea plants caused by Al were not dependent on pH.

| Plant materials and hydroponic culture
Hydroponic culture of tea plants was conducted under ambient light in an unheated greenhouse (120 m 2 ) at Shizuoka University (Shizuoka, Shizuoka, Japan) under an average temperature of 20°C in the spring season (late March to late June) of 2017 and 2018. A slight modification of the culture method described by Konishi et al. (1985) was used. One-year-old rooted tea cuttings of "Yabukita," a leading Japanese green tea cultivar, were transplanted to Wagner pots (1/2000 a; three individuals per pot) containing 12 L tap water adjusted to pH 4.2, and continuously aerated. After 1 week, standard nutrient solutions containing 400 μM Al, prepared from Al 2 (SO 4 ) 3 ‧14-18H 2 O, at pH 4.2 (Konishi et al., 1985) was supplied stepwise for 1 week each at 1/5, 1/2, and full strength to adapt the plants to the hydroponic system. The following experiments were subsequently performed. An overview of the hydroponic experiments performed in this study is shown in Figure S1.
An initial hydroponic experiment (in spring of 2017) was performed to evaluate broadly the effects of pH and the presence of Al on plant growth. Plants were transferred to nutrient solutions adjusted using H 2 SO 4 to various pH values, namely pH 2. 8, 3.2, 3.8, 4.2, 4.8, 5.2, 5.8, 6.5, and 7.5, with or without 400 μM Al, prepared from Al 2 (SO 4 ) 3 ‧14-18H 2 O. Each experiment was conducted using three biological replicates. The solutions were replaced at 2-day intervals to maintain the pH. After 5 weeks, tea plants were harvested following the methods described by Morita et al. (2008). At harvest, the roots were immersed in water at pH 3.0 (adjusted with H 2 SO 4 ) for 3 min to remove Al absorbed on the root surface. After washing with deionized water, the plants were divided into leaves, stems, and roots, and each part was further separated into new and mature parts: new parts were those that had emerged during treatment, and mature parts were those that were present at the start of treatment. Thus, the growth of new shoots, comprising new leaves and stems, and new roots were evaluated as the growing parts. Each sample was weighed fresh, then freeze-dried and re-weighed to determine the dry weight (DW).

| Mineral quantification
Fine powder (50 mg) of freeze-dried samples was digested in 2 ml of 60% HNO 3 at 110°C in DigiTUBE® tubes (SCP SCIENCE, Québec, Canada) for approximately 2 hr. Once the samples had cooled, 2 ml of 60% HClO was added and the samples were heated at 110°C for a further approximately 2 hr. Once digestion was completed, the samples were cooled and made up to a volume of 10 ml with ultrapure water. The total concentration of the following 13 elements was measured, based on selected specific wavelengths using an Total carbon (C) and N were measured by dry combustion using a Vario MAX cube (Elementar, Hanau, Germany) with aspartic acid as a standard.

| Statistical analyses
Plant mineral status was evaluated as mineral content (mg/g DW) and mineral accumulation (mg/plant). Mineral accumulation (mg/ plant) was calculated from the mineral content (mg/g DW) and the plant tissues dry weight (g DW). Significant differences in growth, mineral contents, and mineral accumulation between pH and Al treatments and among pH values were determined using two-way analysis of variance (ANOVA) and simple linear regression, respectively. Significance of correlations between Al and other minerals was determined using Pearson correlation analysis, while correcting for multiple comparisons. The q-values were calculated for multiple testing using the Benjamini-Hochberg false discovery rate (Benjamini & Yosef, 1995) from the p-values obtained in the correlation analysis, performed using the "corr.test" function of the R package "psych" ver. 1.9.12.31 (Revelle, 2020). The q-values < .05 were considered significant. The data in the figures are the mean ± SD of three biological replicates.
The individual values of each treatment were used for multivariate analysis of the ionome, quantifying the data for 14 elements without Al. Data were normalized by calculating z-score values for each mineral in principal component analysis (PCA). The PCA was performed using the R function "prcomp," and the principal component scores and biplots were plotted using the R package "ggplot2" PERMANOVA was performed using the "adonis" function of the R package "vegan" ver. 2.5-6 (Oksanen et al., 2019).

| Growth in different pH and Al treatments
We evaluated the effects of pH and Al on growth using 1-year-old rooted tea cuttings ( Figure S1). The development of new shoots was observed only at pH 3.2, 3.8, and 4.2 with Al treatment (+Al), and there was no shoot growth without Al treatment (−Al) (Figure 1a

| Al content and accumulation in response to pH treatment
We analyzed the Al content and accumulation in new leaves, mature leaves, and new roots, as the predominant tissues for mineral accumulation in tea plants, under different pH conditions. The Al content in the three tissues was increased by Al treatment (Figure 3a

| Tissue ionome dynamics in response to pH and Al treatment
To determine the effect of pH and Al on the distribution of a range of elements in the predominant mineral-accumulating tissues, we used ICP-OES and a CN analyzer to analyze the following 14 elements, in addition to Al, constituting the ionome: Fe, Na, B, P, S, Si, Ca, Cu, K, Mg, Mn, Zn, N, and C (Table S1). PERMANOVA revealed that the ionome profiles in all the three tissues tested were significantly affected by pH and Al treatment (Table 1). In the PERMANOVA analysis, pH treatment explained 7.2%, 10.6%, and 8.0%, whereas Al treatment explained 14.8%, 16.7%, and 54.3% of the total variability in new leaves, mature leaves, and new roots, respectively (Table 1). PCA showed a clear separation of ionome profiles not by pH treatment but by the presence/absence of Al in all the three tissues tested, and loading factor biplots identified the elements that contributed to that separation (Figure 4). In new leaves, the difference in ionome profiles with Al treatment was observed as the second principal component (PC2; Figure 4a), representing 19.6% of the total variation. The predominant elements that contributed to PC2 were Mn, B, Si, and Fe (Figure 4a). In mature leaves, the difference in ionome profiles with Al treatment was also observed

| Correlation between Al and other minerals
Correlation analyses of the ionome dataset revealed positive and negative correlations among the 15 elements in each of the three mineral-accumulating tissues ( Figure S3). To understand the relationship between the contents of Al and each mineral, we focused the TA B L E 1 Statistical tests by permutational analysis of variance (PERMANOVA) for the ionome profile under acidic pH levels with and without Al analysis to correlations between Al and the other minerals ( Figure 6).
The minerals correlated with Al were differed in each of the three tissues tested but some similarities were observed, as follows. Na was positively correlated with Al in both new leaves and new roots ( Figure 6). B and Mn were positively correlated with Al in mature leaves and new roots ( Figure 6). Si was negatively correlated with Al in the three tissues. Ca and Fe were negatively correlated with Al in new leaves and new roots ( Figure 6).

| D ISCUSS I ON
The major rhizotoxicity factor in acid soils is the excess of H + and Al 3+ , which causes inhibition of root growth and nutrient uptake (Kochian et al., 2004). However, tea plants can grow vigorously in acidic soil. Furthermore, in the presence of Al, the growth of tea plants is not inhibited but rather stimulated (Ghanati et al., 2005;Konishi et al., 1985;Morita et al., 2008;Sun et al., 2020). This phenomenon was observed in the present study (Figures 1 and 2), with our results confirming that Al was beneficial to the growth of tea plants.
Hydroponic experiments under diverse acid pH conditions revealed that tea plants grew well at pH 4.0-5.4, and especially around pH 4.2. Although tea plants were able to survive under severe acidic conditions, such as pH less than 4.0, growth was inferior to that at the optimum pH 4.2. However, at pH 2.8-3.0, tea plants did not grow even in the presence of Al, and new leaves and roots did not develop or grow (Figure 1). In some Japanese tea fields, heavy application of N fertilizer has caused severe soil acidification to around pH 3.0 (Tokuda & Hayatsu, 2004). Tachibana et al. (1995) reported that the soil pH of 126 tea fields in Mie Prefecture, a major Japanese green tea cultivation region in Japan, ranged from pH 2.9 to 5.9, with that of most fields less than pH 4.0. The present results indicate that the soil pH is important for improvement of tea production and quality.
The optimum pH for the growth of tea plants in hydroponic culture was around pH 4.2, with inferior growth under pH less than 3.8 or higher than 5.0. In addition, under the optimum pH conditions, the growth stimulation by Al was pronounced (Figures 1 and 2).
The Al content and accumulation were increased by Al treatment, and this was observed especially under the optimal conditions for the growth of tea plants around pH 4.2 (Figure 3). These results suggest that tea plants actively absorbed and accumulated Al under the optimum acidic pH for growth. In new roots, the Al content and accumulation were affected by pH and declined with decrease in pH ( Figure 3c). These results suggest that excessive H + competitively inhibited Al uptake in the roots of tea plants.
Ionomics is a useful tool to understand changes in physiological processes in response to nutrient status . The alteration to ionome dynamics was confirmed in response to environmental factors (Chu et al., 2016;Quadir et al., 2011;Wu et al., 2013).
In acidic soil, Al affects the cellular homeostasis of various ions, resulting in the inhibition of plant growth (Babourina & Rengel, 2009;Bose et al., 2010aBose et al., , 2010bBose et al., , 2011Bose et al., , 2013Plieth et al., 1999;Rengel & Zhang, 2003). However, few studies have investigated the effect of Al as a TA B L E 2 Statistical test results by two-way ANOVA results for the ionome data under acidic pH levels with and without Al  Table 2 and Table S1).
It has been considered that the activity of these cationic nutrients at the cell membrane surface might compete with excess Al 3+ (Kinraide et al., 1992). However, in leaves the contents of these elements were not affected (Figure 4a,b, Table 2 and Table S1). These results suggest that Al suppressed the absorption of many cationic elements, but in its beneficial role it might aid their efficient translocation from the root to the shoot in tea plants. Therefore, Al might complement the nutrient functions of these cationic elements and enable good growth in poor nutrient environments such as acid soils. However, to clarify the possible nutritional roles of Al in tea plants, further physiological experiments are needed.
Interestingly, the only element significantly increased in content by Al treatment was Mn, despite it being a cationic nutrient; Mn content was not affected by pH conditions, and an Al-induced increase in Mn accumulation in the leaves was also observed ( Figure 5). In rice, which is the most tolerant to Al among the small-grained cereal crops (Foy, 1998), Al alleviated Mn toxicity, which was attributed to decreased shoot Mn accumulation resulting from an Al-induced decrease in root symplastic Mn uptake . This phenomenon of Al-induced decrease in Mn uptake has been observed in other plant species (Blair & Taylor, 1997;Clark, 1977;Taylor, Blamey, & Edwards, 1998;Yang, You, & Xu, 2009). The decrease in root symplastic Mn uptake results from an Al-induced change in cell membrane potential according to the Gouy-Chapman-Stern model (Kinraide, Yermiyahu, & Rytwo, 1998;Kopittke, Wang, Menzies, Naidu, & Kinraide, 2014;Wang et al., 2015). Therefore, in tea plants, in contrast to other plant species, Al might enhance Mn uptake and translocation via a Mn transporter, unlike the decrease in Mn electrical activity in the cell membrane induced by Al. Given that toxicity caused by excess Mn occurs in acidic soils in the same manner as Al toxicity (Kochian et al., 2004), both Al and Mn stress-adaptation mechanisms might develop simultaneously in acidic soils through coordinated adaptation processes.
To understand the mechanism of adaptation to acidic soils, further verification of the interaction between these two mineral stresses is a topic for future research.
Correlation analysis confirmed the results revealed by PCA ( Figure 6). B and Mn were positively correlated with Al in both mature leaves and new roots, which are the predominant Alaccumulating tissues in tea plants ( Figure 6). Hajiboland et al. (2015) reported Al-induced increases in the contents of B in the root cell wall (CW) and of CW-bound phenolic acids, but not of lignin, and suggested that increased B partitioning to the CW and reduced lignification were important components in the growth F I G U R E 6 Correlation coefficient between Al and other minerals in the predominant mineral-accumulating tissues of tea plants.
Asterisks indicate significant correlations with Al. Significance of correlation between Al and other minerals was determined by statistical test for correlation correcting for multiple testing using the Benjamini-Hochberg false discovery rate (BH-FDR, q < 0.05)

F I G U R E 7
Overview of the Alresponsive tissue ionome in tea plants stimulation by Al. The present analysis also confirmed interactions between Al and B.
In conclusion, we revealed that the growth of tea plants was stimulated by both Al and acidic pH, with optimum growth observed in a narrow range around pH 4.2 and inferior growth at pH less than 3.8 or higher than 5.0. Under the optimum pH conditions, Al markedly stimulated growth and Al accumulation at the whole-plant scale. Furthermore, we showed that the alteration to ionome profiles caused by Al in tea plants did not depend on pH ( Figure 7). Our findings indicated that the distinct alterations in tissue ionome in tea plants were possibly attributable to the development of adaptations to acid soils. Through integration of the present results with other omics data, such as genome, transcriptome, and metabolome data, and use of phenotypes associated with genetic variation, these findings will accelerate progress in understanding the roles of Al as a beneficial element for some species, such as tea plants, that are well adapted to acid soils. Recently, the draft genomes of the two important tea varieties, C. sinensis var. sinensis (Wei et al., 2018) and var. assamica (Xia et al., 2017), were sequenced using a next-generation sequencing platform.
Next-generation sequencing technologies are accelerating the application of transcriptome analysis in tea plants; for example, use of RNA-sequencing enables advances in elucidation of various environmental responses in different genotypes (Bai et al., 2019;Li et al., 2015;Li, Xiang, et al., 2017;Li, Huang, et al., 2017;Lu et al., 2018). Recently, Li, Huang, et al. (2017) reported an Al-responsive de novo RNA-sequencing transcriptome analysis of tea roots that indicated common and distinct Al-tolerance mechanisms between tea plants and rice, Arabidopsis, and buckwheat. The present findings provide a foundation for the nutritional knowledge needed to clarify the role of Al as a beneficial.

ACK N OWLED G M ENTS
This work was supported by the Japanese Society for the Promotion of Science Grant-in-Aid for Scientific Research, number 20H02886 (T.I.). We thank Huw Tyson, PhD, from Edanz Group (www.edanz editi ng.com/ac) for editing a draft of this manuscript.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest associated with this manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that supports the findings of this study are included in the article and supplementary material.