The citrus fruit trees are superbly adapted to the acid soil with potential high Al in south China . Understanding of Al partition and mobilization in vivo is pivotal to reveal the Al tolerance of citrus species. Besides, it is of great significance to disclose the Al binding site of citrus species for Al toxicity mitigation. The present study addressed both challenges. The hydroponic culture had been widely carried out to explore the ion behavior of citrus species [24, 25]. Compared to our previous study in sandy culture with 1.0 mM Al treated for 18 weeks , the present 21 days’ hydroponic culture of citrus species resulted in almost the same Al level in leaves, indicating a reliable treatment of the study.
It has been evidenced that Al-induced phytotoxicity has many target sites from the apoplast to symplast in higher plants . Accordingly, plant species varied in Al-tolerance have evolved different strategies to cope with Al toxicity based on Al distribution and translocation. Plant species native to acid soils are often found to retain excess Al in insensitive roots, protecting leaves from metabolic disruption . For instance, Kopittke et al.  reported that the Al-tolerant wheat accumulated more than four times of Al in roots compared to the sensitive line. Similarly, the higher Al content on the root apex was also observed in Al-tolerant common bean compared to the Al-sensitive genotype . The present results supported higher Al in roots under Al stress compared to shoots in citrus species (Fig. 1). Besides, C. sinensis had a significantly higher Al content in lateral roots but significantly lower Al content in shoots than C. grandis under Al stress. Thus, the less Al translocation from roots to shoots in C. sinensis might contribute to the mitigation of Al-toxicity in the shoot. Likewise, the higher Al translocation of C. grandis than C. sinensis was also reported in our previous study by 1.0 mM Al stress under 18 weeks’ sandy culture . However, the Al stress within 21 days did not significantly differ in the biomass accumulation of two citrus species (data not shown). With the stress duration increased to 15 weeks, the C. sinensis seedlings had remarkably higher biomass accumulation than C. grandis in both leaves and roots (Fig. s1). Conclusively, the relatively higher Al tolerance of C. sinensis is related to a less Al translocation from the roots to shoots.
Plant root cell wall is the first defense conferring Al toxicity. Clarkson et al.  revealed that over 85% of Al accumulated on the cell wall of barley roots. For woody plants, more than 88% of total Al was localized in the root cell wall of the conifer . In the study, the cell wall of citrus lateral roots accumulates higher Al content than the cell organelle and cytoplasmic supernatant. The ratio of Al concentration on the cell wall, cell organelle and cytoplasmic supernatant is about 8:2:1 (Fig. 2a) under 1.0 mM Al stress, indicating the prominent roles of the root cell wall in Al immobilization of citrus species. Interestingly, the ratio of cell wall-binding Al is very close to the finding of Al-treated tea (Camellia sinensis) roots . Moreover, the Al distribution at cell wall fraction is in the order of pectin > HC-II > HC-I > cellulose, suggesting the pectin holds the most while the cellulose has the least Al. The contribution of pectin in Al sequestration was also reported in rice roots . Li et al.  proposed that a high density of carboxylic groups on the pectin contributes to Al binding. Further studies regarding pectin content and related structural deformation under Al stress of citrus species are needed to reveal the role of pectin in Al detoxification.
Ma et al.  reported that the cell wall of Al sensitive wheat had higher Al retention than Al tolerant cultivar under 10 µM Al within 9 hours’ duration. By contrast, we observe that dry roots of C. sinensis had a remarkably higher Al content on the cell wall than that of C. grandis (Fig. 3), which is consistent with the higher Al content of lateral roots in C. sinensis than C. grandis. Therefore, we propose that the Al distribution pattern in higher plants depends on the toxic intensity, such as Al level and stress duration. For example, the Al tolerant cultivar might exclude Al encountering weak Al stress, which resulted in less Al accumulation. However, when the Al exclusion is not enough for Al detoxification, the mechanism of Al translocation in vivo was activated, such as Al stabilization on the roots or cell wall. For another, the Al distribution and translocation might reflect the flexible strategies for Al detoxification between woody and gramineous plants considering the different root structures and extreme variation of root biomass.
The adsorption and desorption kinetics demonstrated that the root cell wall of C. sinensis, an Al-tolerant species, had a higher Al affinity than C. grandis (Fig. 5). By contrast, the root cell wall of C. grandis exhibited a lower Al adsorption and a higher Al desorption, indicating less tight Al-binding on the root cell wall, which would facilitate higher Al translocation from apoplast to symplast. Therefore, we infer that Al-tolerant woody plants were prone to retain excess Al on the root cell wall to diminish Al translocation owing to their high capacity of the root systems. Besides, the Al binding firmly on roots is economical for Al resistance considering the energy cost during Al translocation. The findings of the present results also implied that organic material prepared from cell walls is promising in alleviating the Al toxicity of the citrus plants in acidic red soils.
The Al binding resulted in modification of the root cell wall, which could be assessed by FTIR analysis [35, 36]. In the study, the results that almost no new characteristic peak emerged indicated less effect of Al toxicity on the types of functional groups on the cell wall by Al toxicity overall in two citrus species. The modification of cell wall by Al stress might mainly be dependent on the abundance of chemical groups on the root cell wall of citrus species. For instance, the spectra at 3400 cm− 1 (-OH stretching), was shifted to 3396 cm− 1 under Al stress in C. grandis, suggesting the changed hydrogen-bonding mode and the destroyed connection of cell wall components by Al toxicity (Table 1). The results approved less Al tolerance of C. grandis than C. sinensis by considering the flexible deformation of hydrogen bonds between molecules . Also, a study indicated the absorbance at 1740 and 1649 cm− 1 represents the absorption of the esterified and non-esterified carboxyl groups of pectin, respectively . The present results of downregulated relative absorbance at 1740 cm− 1 and 1649 cm− 1 were coincident with significantly higher Al accumulation in the pectin (Fig. 6c), suggesting the role of cell wall pectin in Al-binding under Al toxicity. Besides, it is also interesting to find that the vibrations from 1200 cm− 1 to 900 cm− 1 (Table 1), which belong to the polysaccharide fingerprint region , shifted and decreased under Al toxicity. The results indicated that the altered structure and content of cell wall polysaccharides under Al toxicity would affect the Al binding on the root of citrus species. Both of the digital subtraction spectra (Fig. 6c) and the OPLS-DA (Fig. 6c) of relative absorbance in two citrus species supported a much apparent alteration of the cell wall in C. grandis compared to C. sinensis, such as severer damage under Al toxicity. Similarly, a higher relative absorbance of the upper leaves corresponding to a much obvious symptom of boron deficient orange seedlings compared to lower leaves was also reported based on the FTIR analysis . Further studies based on isotope labeling of Al and pectin deformation and polysaccharides quantification of the citrus root cell wall are needed to disclose the Al spatial and temporal distribution.