3.1 An Al-tolerant Eucalyptus hybrid clone has enhanced accumulation and exudation of malate and citrate
The available evidence indicates that the Al-induced secretion of organic acids from roots may lead to the detoxification of Al in higher plants (Ma, 2000, 2005). A role for organic acids leading in Al tolerance in Eucalyptus has been observed previously (Nguyen et al., 2003; Silva et al., 2004; Tahara et al., 2008). The lower root concentration of Al coupled with the higher root secretion of citrate and malate in Al-tolerant E. grandis × E. urophylla clone G9 than that in Al-sensitive E. urophylla clone W4 suggested that secretion of these two organic acids was involved in increased tolerance to Al in G9. This trait was consistent with the report in Al-tolerant E. camaldulensis (Nguyen et al, 2003; Tahara et al., 2008; Ikka et al., 2013). Tahara et al. (2008) documented with E. camaldulensis that citrate showed the strongest Al-binding capacity among citrate, oxalate, malate and phosphate. It was likely that the Al-stimulated accumulation and secretion of citrate may be the main underlying mechanism contributing to detoxification of Al in Eucalyptus roots, particularly in Al-tolerant genotypes. However, Silva et al. (2004) put forward the hypothesis that Al tolerance was due to the internal detoxification of Al by malate complexation. These conflicting results made the role of malate in conferring Al tolerance on Eucalyptus unclear. Adding to the complexity, Ikka et al (2013) inferred that the types of organic acids in response to Al may vary among Eucalyptus species. The features of malate and citrate accumulation and secretion in Al-tolerant hybrid clone G9 and Al-sensitive parental clone W4 have provided clues for further exploring Al-induced genes or proteins.
3.2 The transport pathways of citrate and malate secretion are potentially regulated by carrier proteins or an anion channel
The rapid release of organic acid in response to exposure to Al suggested that pre-existing anion transporters on the plasma membrane quickly initiated organic acid secretion without the need to produce new proteins, but a lag in release suggests a requirement for relevant gene expression and / or protein synthesis (Ma, 2000; Kollmeier et al.,2001; Ryan et al., 2003). Clone G9 had no significant delay in the malate secretion followed by later citrate secretion, while W4 had a significant lag in malate secretion and did not activate citrate secretion. On the other hand, both PG and CHM significantly reduced the Al-induced secretion and internal concentration of citrate in both Eucalyptus clones roots as well as the malate in G9 roots, but CHM had no impact on malate secretion in W4 root, which may indicate that there is genetic variation in this response within the genus. Generally, Al-tolerant species or varieties had higher activation and quantities of carrier proteins and anion channel proteins than Al-sensitive genotypes (Piñeros et al., 2002; Li et al., 2009; Yang et al., 2011; Yu et al., 2012). Therefore, it may be inferred that the citrate and malate secreted by the Al-tolerant G9 may require both an anion channel and synthesis of new proteins. Generally, if protein synthesis is required for the production of a carrier protein, then one would expect an obvious lag of several hours before secretion becomes apparent, as observed for the putative citrate channel in G9. However, the secretion of malate by G9 rapidly increased after exposure to Al, suggesting there could be other factors responsible for the rapid secretion of malate in Al-tolerant genotypes.
The beneficial effect of Al on growth of plants adapted to low pH soils was ascribed to increased uptake of nutrients by roots (Osaki et al, 1997). Similarly, Al tolerance in Eucalyptus was explained by the maintenance of nutrients and photosynthesis (Silva et al, 2010, 2017). Another study found that a new low-molecular-weight Al-binding ligand, oenothein b, from roots contributed to Al tolerance in E. camaldulensis (Tahara et al., 2008, 2017), encouraging further exploration to find other mechanisms that help fast-growing Eucalyptus clones to manage excessive soluble Al in acidic soil. Thus, further work is necessary for a wider understanding of other factors that might be responsible for Al tolerance in hybrid Eucalyptus clones and how associated molecular mechanisms might be activated through hybridization.
3.3 The effects of Al on enzyme activities involved in the root metabolism of citrate and malate
The Al-induced decrease in root internal malate concentration in response to the increase in malate secretion in W4 contrasted with the increase in both internal root concentration and secretion of malate in G9. This contrast suggested that there could be another factor involved in the regulation of malate secretion. As in other plants, the secretion and accumulation of citrate and malate in Eucalyptus were closely linked with changes in the activities of several enzymes involved in organic acid metabolism. We observed that in both clones CS and PEPC activities were markedly induced by Al, while ME activity significantly decreased. In G9, ACO and MDH activities were dramatically decreased in the presence of Al, while IDH activity was unaffected. In contrast, in W4, ACO and IDH activities were significantly increased by Al exposure, while MDH activity was unchanged. Apparently, the accumulation and secretion of citrate in G9 was achieved by stimulating CS activity and suppressing citrate catabolism. Citrate accumulation in G9 may have been supported by decreased flux through ACO. Previous studies also found increased CS activity and decreased ACO activity were linked with accumulation and secretion of citrate from roots of Al-tolerant species, such as rye (Li et al., 2000), Paraserianthes facataria (Osawa and Kojima, 2006), and soybean (Xu et al., 2010). However, Ikka et al. (2013) reported that the Al-induced increase in citrate concentration in roots of E. camaldulensis was not caused by a change in CS activity, but only by reduced ACO activity to suppress citrate catabolism. The increased activity of PEPC in both clones upon exposure to Al may contribute to the increased biosynthesis of organic acids by feeding oxaloacetate carbon skeletons into the TCA cycle (Dong et al., 2004). In G9, the apparent synergistic effect of PEPC and CS on the synthesis of citrate was greater than that of other enzymes, such as ACO and IDH, on citrate catabolism, resulting in the production and secretion of more citrate. In the case of W4, the increased ME activity may have been responsible for the lower malate accumulation in this clone. Both PG and CHM seemed likely to enhance the activity of enzymes that were beneficial to citrate synthesis and decreased the activity of enzymes that metabolized citrate. The Al-induced synthesis or catabolism of citrate and malate was regulated by the balance among the various organic acid-metabolizing enzymes, which served to produce differential secretion of these two organic acids in Eucalyptus clones differing in their Al tolerance.