A biochemical mechanism used by plants to cope with Al stress involves the activation of membrane transporters responsible for organic acids secretion from the root apex to the rhizosphere. These organic acids form non-phytotoxic stable complexes with Al3+, preventing its absorption by the roots [4, 26, 27]. The ALMTs proteins are among the transporters that play pivotal roles in the adaptation to acid soils. These proteins exudate malate to the rhizosphere in the presence of Al, conferring tolerance, via chelation of Al3+. To the best of our knowledge, ALMT gene family members were not identified in sugarcane (Saccharum spp.) to date. Genome-wide analysis of the recently released sugarcane genome  identified 11 ALMT genes in sugarcane, which were phylogenetically divided into 4 different clades (Fig. 1). The study of the expression pattern of these different ALMT genes confirmed the involvement of the identified transporters in Al responses in sugarcane, since high levels of SoALMT transcripts was observed in roots of NT plants exposed to the metal (Fig. 6).
However, the expression of SoALMTs 1, 3, 6, 8 and 10 were not observed in roots of sugarcane in our experimental conditions, possibly because these genes are expressed in other tissues that were not studied in this work. ALMT proteins are also known to regulate several physiological responses in plants such as guard cell regulation, anion homeostasis, fruit quality, seed development and microbe signaling network . Therefore, these transporters are present and expressed in different plant tissues and different developmental stages.
In sorghum (Sorghum bicolor), a membrane transporter gene belonging to the multidrug and toxic compound extrusion (MATE) family was identified and characterized as an Al-activated citrate transporter gene responsible for the Al-tolerance in this crop, and the overexpression of SbMATE conferred tolerance in Arabidopsis plants . Moreover, the overexpression of close homolog of SbMATE, the Brachypodim distachyon MATE gene (BdMATE), in Setaria viridis conferred tolerance to Al. S. viridis is a C4 plant that is emerging as a model for grasses [3, 29]. Based on these studies, transgenic sugarcane lines constitutively overexpressing the SbMATE gene were generated to verify if the transgenic plants could demonstrate increased tolerance to Al. Sugarcane RB855156 was successfully transformed to overexpress the SbMATE gene driven by ZmUbi1 promoter using a protocol developed by our group . Seventeen independent transgenic events were generated and screened for Al tolerance in plants growing in a hydroponic system (Additional file 3: Fig S3), supplemented with an established concentration of Al3+ activity. Two out of the seventeen events demonstrated significant sustained root growth under Al treatment when compared to NT plants and used for further detailed analysis.
In hydroponic conditions, roots from both NT and transgenic plants grown in Hoagland’s solution in the absence of Al developed a brownish coloration after 2 weeks (Fig. 3), which appears to be correspondent to oxidative damage or accumulation of phenolic compounds . Interestingly, in the presence of Al, sugarcane roots became vigorous with decreased symptoms of oxidative stress, indicating that the cultivar used for our studies is, at least to some extent, tolerant to Al. However, NT plants were unable to sustain root growth in the presence of Al over the period of the experiment, while the 2 transgenic events tested were able to maintain root growth (Fig. 4), in addition to increased number of adventitious roots (Fig. 3), indicating that SbMATE plants might be more tolerant to the metal when compared to NT plants. In addition, hematoxylin staining revealed that transgenic sugarcane roots did not accumulate Al in their apex, as indicated by lack of the purple coloration typical of the interaction between Al and the dye (Fig. 5a). It is known from previous studies that commonalities exist between Al and oxidative stress-induced gene expression in sugarcane apical roots, with several antioxidant genes upregulated under Al stress . The high levels of antioxidant gene expression under Al treatment could explain the loss of oxidative damage symptoms observed in roots of sugarcane RB855156 after Al treatment. Moreover, commercial cultivars of sugarcane are generally regarded as tolerant to Al, due to extensive breeding that has culminated with modern cultivars such as RB855156 [13, 32, 33]. As discussed by Guo et al. (2017) , which demonstrated root adaptive responses to different Al-treated Citrus cultivars, other factors could be responsible for Al-tolerance in plants, in addition to the antioxidant capacity. These factors include higher external Al detoxification capacity via enhanced Al-induced secretion of organic acid anions, a more efficient chelation system in roots, higher capacity to maintain the cellular phosphorus homeostasis by enhancing phosphorus acquisition and utilization, higher adaptive responses to Al concerning cell wall, cytoskeleton and carbohydrate metabolism and upregulation of genes related to fatty acid and amino acid metabolism . However, despite the high tolerance of sugarcane to Al, the extent and severity of soil acidification after intensive cultivation indicates that even slight susceptibility to the metal may result in severe economic losses . Thus, the development of varieties with improved tolerance to Al is pivotal to ensure a suitable harvest.
Organic acids (OAs) secretion from the root apex to the rhizosphere is an important mechanism used by plants to cope with Al stress as OAs form non-phytotoxic stable complexes with Al3+, preventing its absorption by the roots . However, OAs are also important components of plant primary metabolism. Malate, fumarate, lactate and citrate, produced via tricarboxylic acid pathway (TCA), are among organic acids of fundamental importance for several biochemical pathways, including energy production, formation of precursors for amino acid biosynthesis and in modulating adaptation to the environment at the whole plant level [7, 35]. In this context, a balance between the positive effects of OA release and the disadvantage of losing valuable carbon sources is a desirable feature in the selection of transgenic plants constitutively expressing transporters involved in exudation of OAs. Thus, the transcription levels of several Al-responsive genes in roots of sugarcane in the presence or absence of the metal, including genes encoding intermediate enzymes of the TCA pathway, such as citrate synthase (CYS), malate dehydrogenase (MDH) and fumarate dehydrogenase (FUM) was investigated. First, these genes were identified in the sugarcane genome to perform qRT-PCR analysis to determine their expression levels in hydroponically grown NT or transgenic plants, submitted or not to Al stress (Fig. 7a). As expected, SbMATE plants showed higher levels of SoCYS expression even in the absence of Al, possibly due to the increased concentration of citrate exudation.
In the presence of Al, roots of both NT and transgenic plants increased their SoCYS transcription levels, indicating that citrate production is involved in Al stress responses in sugarcane. Malate dehydrogenase (SoMDH) gene expression levels were drastically increased in roots of NT and transgenic plants submitted to Al when compared with hydroponically grown plants in the absence of the metal, suggesting the involvement of malate in sugarcane responses to Al. These results corroborate with the high transcription levels of SoALMTs verified in roots of sugarcane submitted to Al (Fig. 6). Interestingly, higher malate exudation was found in transgenic plants in comparison to control in the presence of Al (Fig. 5b). It was found that citrate exudation is accompanied by malate efflux in transgenic events, possibly indicating a biochemical compensatory mechanism of organic acids in transgenic plants. Fumarate dehydrogenase (SoFUM) gene expression levels increased in roots of NT plants submitted to Al, but it was not significantly altered in roots of transgenic plants under the stress. These data demonstrate that sugarcane overexpressing SbMATE might be using TCA pathway intermediates in a greater extent compared to NT plants. Indeed, increased Al resistance correlates with higher rates of citrate and malate exudation in several plant species, as observed for snapbean, maize and Cassia tora [36, 37, 38, 39] and it appears to be the case also in sugarcane (Fig. 5b). Moreover, alternative glycolytic pathway genes were also differentially expressed in Al-treated roots of two Citrus cultivars, which demonstrate differential responses to Al and phosphorous . These results reinforce that glycolytic pathways are actively involved in Al responses in different plant species. It is worth mentioning that the increase of organic acid secretion is not always the main mechanism for Al-tolerance in plants. For instance, phosphorus (P) supply can alleviate Al-toxicity through increasing immobilization of Al in roots and P levels in seedlings rather than through increasing of OA anion secretion in Citrus . In this regard, it is important to verify soil conditions to improve Al-tolerance in different plant species.
Finally, the expression pattern of some genes known to be associated with Al responses was also investigated. The orthologous genes for STOP1, STAR1 and NRAT1 were identified in the sugarcane genome and their transcription levels were investigated as described above for the TCA pathway genes. STOP1 is a zinc-finger transcription factor that co-regulates a key gene in Al tolerance mechanism in Arabidopsis and appeared to be required for AtMATE expression and Al-activated citrate exudation . It is known that the transcription regulation exerted by STOP1 can be activated not only by Al, but also by low pH . In rice studies, Arenhart et al. (2014)  found that STAR1 gene was the only ABC gene whose transcription level was increased in the Al-treated NT plants, but decreased in the ASR5_RNAi transgenic plants compared to the untreated NT plants. STAR1 was one of the ASR5 target genes identified in the ChIP-Seq analysis, and ASR5 binding to the STAR1 promoter region was confirmed via in vitro DNA-binding assays . This disruption resulted in hypersensitivity to Al toxicity . NRAT1 is a plasma membrane Al3+ transporter located in root apical cells and responsible for Al tolerance in rice. As demonstrated by Xia et al. (2010) , knockout of NRAT1 resulted in decreased Al uptake, increased Al binding to cell wall, and enhanced Al sensitivity. The expression of NRAT1 is up-regulated by Al in the roots and regulated by a C2H2 zinc finger transcription factor (ART1) in rice, and this mechanism is required for a prior step of final Al detoxification through sequestration of Al into vacuoles . The transcription levels of SoSTOP1, SoSTAR1 and SoNRAT1 were drastically increased in roots of sugarcane submitted to Al treatment, suggesting their involvement in Al tolerance pathways in sugarcane.
Unfortunately, hydroponically grown sugarcane does not achieve developmental stages where important measurements such as sucrose content or biomass can be performed. These measurements are of fundamental importance to verify if SbMATE plants are suitable for agricultural purposes. Field trials performed in the Cerrado region of Brazil, using these candidate elite events, are currently underway to address these questions. It is also worth noticing that the detailed mechanism of sugarcane Al-tolerance was not the scope of the present study. The identification of genes possibly involved in Al responses in sugarcane such as SoALMT or TCA cycle genes might help to elucidate the mechanism of Al-tolerance in sugarcane.