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 [32, 33]. A role for organic acids leading to Al tolerance in Eucalyptus has been observed previously [12, 18, 19]. The lower root tip 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 the increased tolerance to Al in G9. This trait was consistent with the results reported for Al-tolerant E. camaldulensis [12, 18, 34]. Tahara et al. [12] documented in E. camaldulensis that citrate had the strongest capacity to bind Al among citrate, oxalate, malate and phosphate. Thus, it was likely that the Al-stimulated accumulation and secretion of citrate was the main underlying mechanism contributing to detoxification of Al by Eucalyptus roots, particularly in Al-tolerant genotypes. However, Silva et al. [19] put forward the hypothesis that Al tolerance was due to the internal detoxification of Al by complexation with malate. These conflicting conclusions left the role of malate in Eucalyptus tolerance unclear. Adding to the complexity, the types of organic acids produced and released in response to Al may vary among Eucalyptus species [34], as do the quantities, as shown here for malate and citrate. The features of malate and citrate accumulation and secretion in Al-tolerant hybrid clone G9 and Al-sensitive parental clone W4 have provided further clues for the identification Al-induced genes or proteins.
3.2 Newly synthesized carrier proteins involved in citrate secretion, but malate secretion facilitated by a pre-existing anion channel in E. grandis × E. urophylla
A rapid release of organic acid in response to exposure to Al would suggest that pre-existing anion transporters on the plasma membrane quickly initiated organic acid secretion without the need to produce new proteins; however, a lag in the release of organic acids could indicate that gene expression and/or protein synthesis was required [33, 35, 36]. There was no significant delay in malate secretion by G9, followed by an increase in the secretion of citrate after a lag period of more than 3 h. In contrast, in W4, there was a lag of more than an hour after Al exposure before malate secretion became apparent, while Al exposure did not induce the production or secretion of citrate. Thus, 24 h after exposure to Al, the synthesis and secretion of malate and citrate by G9 was much greater than in W4.
Both PG and CHM significantly reduced the Al-induced secretion and internal concentration of citrate in roots of both Eucalyptus clones as well as the malate concentration in G9. However, CHM had no impact on malate secretion in W4, indicating that there are different pathways operating for citrate and malate secretion in response to Al in the two clones. Generally, Al-tolerant species or genotypes had stronger induction and higher quantities of carrier proteins on membranes inside root cells and anion channel proteins on the plasma membrane of root cells, than Al-sensitive genotypes [37, 38]. If organic acid synthesis and transport require the involvement of newly synthesized carrier proteins, an obvious lag of several hours before secretion would be apparent, while pre-existing anion channel proteins would allow organic acids to be secreted out of the root more quickly [39, 40]. Therefore, in G9, it seems likely that pre-existing anion channel proteins facilitated the immediate secretion of malate, while a new carrier protein apparently had to be produced before citrate could be transported out of the roots. Anion channel proteins, such as ALMT and MATE/AACT, are localized to the plasma membrane of root cells and transport their substrates to rapidly facilitate organic acid release at phytotoxic concentrations of Al3+ [41, 42]. Furthermore, numerous genes encoding OA transporters have been found to increase OA secretion and to be involved in Al detoxification [43,44]. Sawaki et al. [1] reported that Al-induced excretion of citrate by E. camaldulensis roots was associated with higher expression of EcMATE on the plasma membrane, and that the ectopic expression of EcMATE in tobacco hairy roots enhanced Al-responsive citrate excretion, providing further insight into the molecular mechanism underlying Al resistance in Eucalyptus and the potential for genetic improvement of Eucalyptus. However, other components remain to be revealed, particularly the new protein-coding genes and their functions in organic acid synthesis and transport. For instance, the delay in citrate secretion found in G9 was likely due to the need to produce new proteins involved in the synthesis and delivery of citric acid. For W4, there was no change in citrate secretion in response to exposure to Al, while malate secretion was delayed and did not reach its maximum level for 6 h. Moreover, CHM had no effect on the secretion of malate, but did inhibit its accumulation, indicating that W4 does not lack the capacity to release malate, but rather was restricted in its ability to produce malate.
Increasing organic acids exudation may not be the only effective way to enhance Al resistance of Eucalyptus. We speculate that other organic substances might be involved in detoxifying Al in some Eucalyptus genotypes. A consequence of Al tolerance in Eucalyptus was the maintenance of nutrients and photosynthesis [17,45]. A new low-molecular-weight Al-binding ligand from roots, oenothein b, contributed to Al tolerance in E. camaldulensis [12,46]. In addition, a number of allelochemicals were detected in E. grandis roots and soil by GC-MS [47,48]. Many of these chemicals are involved in either primary or secondary plant metabolism and plant defense processes [49]. These process may interfere with the secretion of low molecular organic acids, or their products may form complexes with Al. For example, one study found that phenolic compounds could be involved in Al detoxification forming strong complexes with Al ions in the cytoplasm of woody plants including E. viminalis Labill. [50]. In addition, transcriptome analysis has revealed that genes associated with flavonoid and phenylpropanoid biosynthetic pathways have key roles in the response of roots of Cunninghamia lanceolata (lamb.) hook. to Al [51]. All these findings encourage further research to identify compounds and the related genes that confer Al tolerance to hybrid clones of Eucalyptus, including the contributions made by allelopathic compounds and other root exudates.
3.3 Secretion and accumulation of citrate and malate in hybrid clone E. grandis × E. urophylla GL-9 were closely linked with changes in CS and PEPC activities
We observed that CS and PEPC activities in root tips of both clones were markedly induced by Al, while ME activity was significantly decreased. Together, these changes likely contribute to the increased biosynthesis of organic acids by feeding carbon skeletons into the TCA cycle [52]. The balance between synthesis or catabolism of Al-induced citrate and malate was regulated by shifts in activities of various metabolic enzymes that together contributed to accumulation of these organic acids to increase Al tolerance in Eucalyptus. Additionally, the addition of inhibitors (PG and CHM) directly or indirectly caused changes in enzyme activities involved in organic acid metabolism.
In the case of E. urophylla clone W4, decreased ME activity may play a greater role in the lower accumulation of malate upon exposure to Al, since MDH activity was unchanged. Meanwhile, the activities of ACO and IDH were significantly increased by Al exposure, which may underlie the lack of an increase in citrate. In E. grandis × E. urophylla clone G9, increased synthesis and secretion of malate seemed to be supported by decreased ME and MDH activity to prevent malate metabolism. We speculate that genes encoding ME may contribute to increased internal malate and citrate concentrations, leading to exudation of these organic acids to confer higher Al resistance, as in soybean [26]. CS is typically regarded as the main enzyme necessary to increase synthesis and secretion of citrate in roots of Al-tolerant plants, such as rye [53], Paraserianthes facataria [54], and soybean [55]. Moreover, transcript levels specifying CS, ALMT and MATE in the root apex of an Al-tolerant cultivar of alfalfa were higher than in an Al-sensitive cultivar [27]. However, Ikka et al. [34] found that the Al-induced increase in citrate concentration in roots of E. camaldulensis was not due to increased CS activity, but was dependent on reduced ACO activity, which would suppress citrate catabolism. Recently, Teng et al. [56] reported that CS, PEPC and IDH may play important roles in organic acid biosynthesis and degradation in Eucalyptus. Our study indicated that the increased synthesis and secretion of citrate that contributed to increase Al-tolerance in E. grandis × E. urophylla was likely achieved by increasing the activities of PEPC and CS, and decreasing the activity of ACO. These three enzymes may be involved in creating the balance between the secretion of malate and citrate in the roots of plants exposed to Al.
From the above, it is clear that key enzymes regulating OA synthesis and exudation vary among of Eucalyptus genotypes. Alterations in the expression of the corresponding genes can affect OA synthesis and exudation resulting in changes in Al tolerance [57]. Some effort has been made in plants to increase the expression of enzymes such as PEPC, CS and MDH by introducing genes encoding these enzymes, for example, in tobacco, alfalfa and canola. Overexpression of these genes would be expected to increase organic acid metabolism and may produce a new citrate synthesis pathway that would contribute to increased Al tolerance in transgenic plants [58-60]. For example, in transgenic canola, overexpression of a CS gene not only led to increased citrate synthesis and exudation, but also changed malate metabolism, which may improve tolerance to Al toxicity [57,61,62]. Since previous studies have indicated that the synthesis of organic acids could be increased by regulating the expression of genes encoding enzymes involved in OA synthesis or transporters involved in OA secretion, transgenic approaches can be expected to provide higher Al tolerance in plants, including Eucalyptus.