High-throughput transcriptomic analysis reveals new players in popcorn (Zea mays) aluminum resistance

Background At the date, the investigation of genes involved in Al resistance has focused mainly in gene expression profiles based on microarray and in short periods of exposure to Al. Under a long term of Al exposure, such as 72 h, resistance maintenance mechanisms might be created in which several metabolic processes are working simultaneously. To investigate new players involved in defense response under Al stress, we tracked the expression profile of two Al-contrasting popcorn inbreed lines during 72 h under Al-stress using Illumina high-throughput mRNA sequencing technology. Results A total of 1,121 differentially expressed genes (DEGs) were identified in the Al-sensitive line and 2,872 DEGs in the Al-resistant line, of which 384 were shared in both lines. Genes categorized in lipid metabolic process and oxidation-reduction were present in both lines, but lipid metabolic process was up-regulated in the Al-resistant line but down-regulated in the Al-sensitive line. The biological process category revealed that the down-regulated genes on the Al-resistant line were involved in signaling. The most significant GO modules were clustered in response to a stimulus. Differentially expressed transporters were also found in both lines, including ABC transporters, Nramp, aquaporins, SWEET transporters, Al-activated malate transporter (ALMT), and multidrug and toxic compound extrusion (MATE). Several transcription factors and genes involved in cell wall modifications were also detected. The Al-resistant line presented genes that play a role in an efficient oxidative system against ROS, involved in cell wall stiffening and dynamic changes of the cell wall to prevent the Al ion transport via the symplast. Also, we detected transporters belonging to families already known to perform a role in Al-detoxification and organic acid exclusion, and we proposed a class of SWEET transporters that might be involved in regulation of vacuolar sugar storage under Al-stress. Although the exact functions of these genes remain to be investigated, these results provide a platform for functional analysis of the defense response against Al-stress in maize.

The most significant GO modules were clustered in response to a stimulus. Differentially expressed transporters were also found in both lines, including ABC transporters, Nramp, aquaporins, SWEET transporters, Al-activated malate transporter (ALMT), and multidrug and toxic compound extrusion (MATE). Several transcription factors and genes involved in cell wall modifications were also detected.

Conclusions
The Al-resistant line presented genes that play a role in an efficient oxidative system against ROS, involved in cell wall stiffening and dynamic changes of the cell wall to prevent the Al ion transport via the symplast. Also, we detected transporters belonging to families already known to perform a role in Al-detoxification and organic acid exclusion, and we proposed a class of SWEET transporters that might be involved in regulation of vacuolar sugar storage under Al-stress. Although the exact functions of these genes remain to be investigated, these results provide a platform for functional analysis of the defense response against Al-stress in maize. Background 3 Aluminum (Al) is the third most abundant element in the earth's crust. In acid soils, with pH values at or below 5, the phytotoxic species Al 3+ is solubilized in soil solution and becomes one of the most important abiotic stresses limiting crop production. Al stress occurs in approximately 30% of world's arable soils and in more than 50% of potentially arable land. From this total, approximately 60% is located in tropical and subtropical regions, and negatively impacting the food supply chain. The phytotoxic form Al 3+ inhibits root growth, thereby altering water and nutrients absorption and consequently reducing plant development [1-3].
Plants use multiple strategies against Al-stress, and two types of mechanisms are described: 1) Alexclusion, preventing the Al entrance in the root apex, and 2) the tolerant mechanism, where Al enters the plant and is detoxified and sequestered [3]. The well-characterized exclusion mechanism is dependent on organic acid (OA) exudation from the root apex [4,5]. Although the root citrate release plays an important mechanism against the Al-stress in maize, as identified from the citrate transporter ZmMATE1 [6], this mechanism is not well correlated with Al-resistance, suggesting that others Al resistance mechanisms are operating in maize roots [7]. Organic acids such as malate, As previously observed, the investigation of Al-responsive genes in maize was focused mainly in an early response. We believe that in a long term of Al exposure, such as 72 h, a robust maintenance mechanism is activated in which several metabolic processes are working at the same time. We investigated new players involved in defense response under Al stress tracked by the expression profile of two popcorn lines (one resistant to and the other sensitive to Al stress) during 72 h under stress by looking for genes that are important for the resistance, and not only signaling. Here we present the first high-throughput RNA sequencing in popcorn and new components regulating the resistance mechanism that will expand knowledge about this trait and might help to identify new markers of Al-resistance in crops.

Results
We generated a range of 38.5 ~ 46.5 million clean reads after the quality process for each sample and obtained around 52.87% of GC content. An average of 80.10% of the reads was mapped, and from this total, only 6.33% presented multiple alignments to the B73 reference genome using the default parameters (Additional file: Table S1).
We observed that the control and +Al treatment were clustered together for both lines, but the lines were grouped into separated clusters, revealing differences on the genetic background between both lines ( Fig. 1) as previously reported by F Rahim, VC Almeida, JMS Viana, C Ribeiro, LA Risso and MP Ribeiro [29]. We detected a total of 1,121 differentially expressed genes (DEGs) for the Al-sensitive line (527 were down-regulated, and 594 were up-regulated) and 2,872 DEGs in the Al-resistant line (1,469 down-regulated and 1,403 up-regulated) at an FDR of q < 0.01 (Fig 2; Additional file: Figure   S1; Additional file: Table S2). The different number of expressed genes in both lines reveal that the Alresistant line has a broader response to Al stress, regulating multiple secondary pathways against the Al damage (Fig. 2). Furthermore, we found 384 common expressed genes in both lines under Al-stress ( Fig. 2; Additional file: Figure S2). Among those, 57 were up-regulated while 51 were down-regulated in both lines. Many of the genes with the same differential expression pattern are involved in response to a stimulus, playing an important role in the defense system. Besides, 145 up-regulated genes in the Al-sensitive line were down-regulated in the Al-resistant line, and many of them are related to "oxidationreduction" class. Meanwhile, 131 genes had the opposite behavior: down-regulated in the sensitive line and up-regulated in the Al-resistant line, and many of them are involved in lipid metabolic process, catalytic activity, metal binding, and oxidoreductase activity ( Fig. 2; Additional file: Table   S3).
The Gene Ontology (GO) analysis categorized DEGs into different groups. For both of the tested lines, many of the differentially expressed genes were classed as belonging to the "metabolic process" category ( Fig. 3; Fig. 4). In the biological process category, "response to stimulus" and "oxidationreduction" were also found highly enriched, "membrane" was enriched in the cellular component category, and "catalytic activity" and "metal ion binding" in molecular function category (Additional files: Figures S3 and S4).
The GO enrichment analysis revealed that genes categorized in lipid metabolic process, oxidation-reduction, oxidoreductase activity, peroxidase activity, and antioxidant activity were present in both lines ( Fig. 3; Fig. 4), but the lipid metabolic process was up-regulated in the Al-resistant line but downregulated in the Al-sensitive line. In the Al-resistant line, we only found significantly up-regulated genes categorized as involved in glutathione transferase (GST) activity (Table 1). Additionally, peroxidase (POD), catalase (CAT), and some reductase (RE) playing a role in reactive oxygen species (ROS) scavenging were detected only in the Al-resistant line, but cytochrome P450 genes with oxidoreductase activity were found in both lines (Additional files: Table S4; Table S5). Among the GST genes expressed in both lines, 11 were up-regulated in the Al-resistant line, contrasting with only three in the Al-sensitive line (Table 1). Down-regulated GST genes were detected: GST34 (Zm00001d029696) and GST U16 (Zm00001d029702) in 11-133 and 11-60, respectively.
The biological process category revealed that the down-regulated genes on the Al-resistant line were involved in signaling. The most significant GO modules were clustered in response to a stimulus. We also detected genes involved in phosphorylation, oxidation-reduction, metabolic sugar process (hexose, fructose, glucose), organic and carboxylic acid metabolic processes, and carbohydrate and lipid metabolic process. However, the up-regulated genes were involved in cellulose, glucan, and suberin biosynthetic processes. We observed only up-regulated genes on cell wall categories in the Alresistant line, which was consistent with the pattern of enriched terms on biological process network analysis conducted in Cytoscape (Additional file: Figure S5). Significant terms found on this analysis were cell wall organization, primary cell wall biogenesis, and polysaccharides responsible for cell wall stiffening (Additional file: Figure S5). On the other hand, GO analysis in the Al-sensitive line identified 30 cell wall enriched genes (Additional file: Figure S3), and no significant enriched terms were reported on protein-protein interaction network, contrasting with 106 genes in the Al-resistant line (Additional file: Figure S5).
The predicted metabolic pathway analysis revealed that the metabolic routes in the Al-resistant line may highly modified under aluminum stress in comparison with the Al-sensitive line (Additional files: Figure S6; Figure S7). After 72 h of Al exposure, this prediction indicated that the brassinosteroid biosynthesis route was induced in the Al-resistant line and deactivated in the sensitive line (Additional files: Figure S6; Figure S7).
Likewise, part of the tricarboxylic acid (TCA) cycle may have been induced in the resistant line. In this predicted map, we visualized a deactivation in 2-oxoglutarate production, and consequently, a reduction in the amino acid metabolism route dependent on this compound. We also observed an exclusive induction of oxaloacetate production in the Al-resistant line. However, in the Al-sensitive line, isocitrate on the TCA cycle was used to produce glyoxylate and induce the glyoxylate cycle. It is noteworthy that the lines showed an opposite pattern when comparing glycine, serine, and threonine metabolism. These pathways were up-regulated in the Al-resistant line and down-regulated in the Alsensitive line (Additional files: Figure S6; Figure S7).
The fatty acid biosynthesis and degradation route may have been induced only in the Al-resistant line (Additional files: Figure S6; Figure S8). We found a curious pattern of alternation in fatty acid biosynthesis between the seven main enzymes. Beta-ketoacyl synthase I, beto-ketoacyl synthase II, fatty acid synthase, fatty acyl-CoA synthase, and 3-hydroxyacyl dehydratase were up-regulated while 3-oxoacyl reductase, fatty acid synthase, fatty acyl-CoA synthase and enoyl reductase (NADPH Sispecific, NADPH and NADH) were down-regulated. However, the final step possibly induced the production of octanoic acid, decanoic acid, dodecanoic acid, tetradecanoic acid and, mainly, hexadecanoic acid. This compound is responsible for inducing cutin, suberine and wax biosynthesis route and might play an important role in cell wall stiffening. In Al-sensitive line, was predicted just an inactivation at the end of the lipid biosynthesis route, probably reducing the production of hexadecanoic acid (Additional files: Figure S7). We also observed that the sphingolipid, linoleic acid, arachidonic acid, and glycerolipid metabolism may have been altered in Al-resistant line, showing an opposite way on the Al-sensitive line.
Differentially expressed transporters were also found in both lines (Table 2; Table 3), including ABC transporters, Nramp, aquaporins, SWEET transporters, Al-activated malate transporter (ALMT), and multidrug and toxic compound extrusion (MATE). The majority of transporters in both lines were ABC transporters, of both the G and B families. The most expressive in the Al-resistant line was the ABC G member 29 and ABC B member 19. Nrat1 was up-regulated, while Nramp6 were down-regulated in the Al-sensitive line. We also found four genes encoding aquaporin proteins only in the Al-resistant line. PIP2-2 (Zm00001d005410 and Zm00001d014285) was up-regulated and TIP3-1 (Zm00001d048520), and an ortholog PIP2-2 (Zm00001d022608), was down-regulated. We also identified seven SWEET transporters, of which one was down-regulated in the Al-resistant line, and three were up-regulated in the Al-sensitive line.
Several transcription factors (TFs) were also detected among our differentially expressed genes. The number of TFs in the Al-sensitive line were 32 and 23 down-and up-regulated, respectively, and the Al-resistant line presented 89 and 38, also down-and up-regulated, respectively (Additional files: Table S6; Table S7). Among these, were found TFs belonged to the families AP2/EREBP, MYB, bHLH and WRKY that were already described in the regulation of gene expression of responsive genes in plants under Al-stress. We found TFs exclusively overexpressed in the Al-resistant line, including ZF-HD, CO-like, ARF, and E2F/DP families.
Four transposable elements were found in both lines (Additional file: Table S2). In Al-sensitive line was found a gene that encodes a retrotransposon protein (Zm00001d029444) and in Al-resistant line was found a gene that encodes a transposon protein CACTA%2C En/Spm sub-class (Zm00001d039524).
Moreover, a transposon protein (Zm00001d041987) was found down-regulated in Al-sensitive, but upregulated in Al-resistant.
To validate the RNA-seq results, thirteen differentially expressed genes were selected and real-time qPCR was performed (Fig. 5). This validation was done with two independent biological replicates (different from the replicates used for the RNA-seq experiment). A total of 84.62% of these selected genes was validated by RT-qPCR.

Discussion
Aluminum toxicity is one of the main factors limiting crop cultivation on acidic soils [30]. For more than 50 years, breeders have explored the genetic diversity to improve Al-resistance in several crops, especially in tropical breeding programs [3]. Some critical Al toxicity events are initiated at the transcriptional, biochemical, and physiological levels [31]. To date, several Al-tolerance mechanisms have been described, but much more needs to be discovered to reveal the complex response to Al-stress. Here, we provided a comprehensive RNA-sequencing analysis revealing new important players involved with Al-resistance in a high concentration of Al 3+ (160 µM) under a long period (72 h).
Our RNA-Seq data revealed a large number of differentially expressed genes regulated by Al-stress in both genotypes. Also, the number of genes differentially expressed in the Al-resistant line was higher than in the Al-sensitive line, suggesting several maintenance responses playing a tolerance role in the Al-resistant genotype. Moreover, under normal conditions, the Al-resistant popcorn has an expression pattern that is clearly distinct from the sensitive genotype, and the basal separation between both popcorn lines by PCA analyses indicates that the resistance is clearly intrinsic to the Al-resistant The predicted metabolic pathway analysis in Al-resistant line, has been showed a probable deactivation of 2-oxoglutarate production from isocitrate in the TCA cycle and induction of oxaloacetate production from the malate (Additional files: Figure S6). This pattern might be due to the release of citrate from maize roots or the production of malate acting as an exclusion mechanism against Al. Citrate release might affect the availability of substrate to increase levels of isocitrate and consequently 2-oxoglutarate. However, further metabolic analysis need to be made to quantify these compounds in popcorn roots. The organization of carboxylic acid metabolism in plants is highly dependent on the metabolic and physiological demands of the cell [36] and the Al-stress condition might induce this non-cyclic flux.
The in silico analysis showed that possibly were no responses detected in the TCA cycle in the Alsensitive line. However, we observed a supposed partial activation in part of the glyoxylate cycle.
Since isocitrate serves as a substrate for activating this cycle (Additional files: Figure S5), this may work as an extra production of succinate and posterior conversion to malate to supply the TCA cycle with metabolites or a new energy source to the cell from acetyl-CoA. To date, no evidence has been presented demonstrating glyoxylate cycle change in plants under Al-stress, and this hypothesis needs further investigation.
Other transporters play an important role in the tolerance mechanism by detoxifying the Al ion inside the cells. The ABC transporters families found in both lines suggest that these transporters work together with other detoxifying systems to increase the tolerance response in Al-resistant line. Nrat1 (Nramp aluminum transporter 1) is a specific Al transporter identified in rice that uptakes Al to cells for sequestration to vacuoles, and it is required for the initial steps of internal Al-detoxification [37].
The best candidate gene as homolog of OsNrat1, showed an up-regulation pattern in Al-sensitive line.
We proposed that, although our sensitive plant can respond with multiple mechanisms against the absorption of Al ion in popcorn roots, this is not sufficient to support this stress due to the metabolic unbalance caused by the Al toxicity condition. Aquaporins are a group of highly conserved membrane proteins that facilitate water transport across biological membranes [38] and, in our study, our observations suggest that tonoplast aquaporins are likely involved in the Al-tolerance response, but it remains unclear whether PIP proteins might play an important role in response to Al-stress in popcorn.
Cellular efflux of sugar plays important roles in the maintenance of sugar efflux in phloem loading, nectar secretion, for the supply such as mycorrhiza, and as maternal efflux for filian tissue development [39]. Plants need to maintain a rigid regulation in the storage and transport of vacuolar sugar to deal with environmental adverse conditions [40]. The role of SWEET in Al-tolerance responses remains unclear, but its play an important role in adverse conditions, such as tolerance to osmotic stress, including cold, high salinity and drought [41] and a mechanism to accumulate sugars in vacuoles as a tolerance mechanism to freezing stress has been described [42]. Based on this, we suggest that these SWEET transporters play a role in maintaining the tight regulation on vacuolar sugar storage.
Plants under aluminum stress might produce ROS in mitochondria, chloroplast, and peroxisome [43] and to defend themselves, they might control the ROS levels by deploying a complex antioxidant In predicted metabolic pathway maps, we reported the induction of fatty acid biosynthesis that may have been produced hexadecanoic acid activating the cutin, suberin, wax biosynthesis route, and down-regulation in part of the fatty acid degradation route in Al-resistant (Supplemental Figure 6).
However, the Al-sensitive line demonstred a predicted down-regulation in the fatty acid biosynthesis pathways (Supplemental Figure 7), demonstrating that fatty acids might contribute to membrane integrity under Al-stress.
Root growth and development response, which are also involved with cell enlargement, were upregulated in the Al-resistant line. Our results indicate that the majority of transcripts associated with hormones were related to cytokinin, auxin (IAA), and abscisic acid (ABA). Abscisic acid might regulate Al resistance in soybean through the possible involvement with citrate release [65]. In Arabidopsis, the AtALMT1 expression levels are up-regulated by ABA, suggesting that this hormone might activate ALMT1 expression as malate transport activity and then inducing organic acid expression [66].
Furthermore, using a microarray approach, Al-stress was shown to induce the expression of ABArelated genes (DREB1A and DREB1A) in A. thaliana [67].
The transcription of Al-tolerance genes might also be activated by IAA [66]. In Arabidopsis mutants with internal Al-detoxification mechanisms, it was verified that endogenous IAA suppress the transport of symplastic Al 3+ to the vacuole, thereby negatively regulating Al-tolerance [68], but exogenous IAA induced high expression levels in AtALMT1, as well as a slight increase in AtMATE expression levels [66].
We also found a class of steroid hormones, the brassinosteroids (BRs), that might minimize the toxic effects caused by aluminum and other heavy metals, thereby reducing the accumulation of these elements due to their capacity to regulate the uptake of ions inside plant cells and inhibit the degradation of lipids resulting from the overproduction of ROS. Furthermore, BRs trigger the accumulation of apoplastic H 2 O 2 , which up-regulates the antioxidant system, thereby increasing the stress tolerance [69][70][71].
Little is known about the effect of BRs in Al stressed plants, but most evidence suggests that BRs improve the response of the antioxidant system. Treating mung bean seedlings with exogenous BRs, 28-homobrassinolide or 24-epibrassinolide (EBL), B Ali, S Hasan, S Hayat, Q Hayat, S Yadav, Q Fariduddin and A Ahmad [72] evidenced an increase in proline content and the activities of SOD, CAT, and guaicol peroxidase in response to Al stress, indirectly contributing to the improvement of plant growth and photosynthesis. Additionally, EBL significantly increased the chlorophyll content and fresh masses of shoots and roots.
In our study, we reported a possible differential response in BRs biosynthesis route between the contrasting lines under aluminum stress for 72 h through in silico metabolic analysis (Supplemental only three down-regulated (bHLH1, OVATE family protein 17, and MYB19). In rice, some authors reported ASR (Abscisic Acid, Stress and Ripening) as involved in Al and other stresses [74]. MYB proteins were found to be regulated under Al-stress in common bean [75] and playing a role in the cleavage mediated by miRNA, essential for responding to Al-stress, in wild soybean [76].
Transcriptional factors belonged to these families were found with different expression regulation in both inbred lines and may be involved in regulation of downstream expression of genes involved in Al stress.
Members of the WRKY family might develop various responses under Al-stress. ZJ Ding, JY Yan, XY Xu, GX Li and SJ Zheng [77] reported that WRKY46 acts as a repressor of expression of AtALMT1 in Arabidopsis. WRKY22 has a positive role in increasing the expression of OsFRDL4, a gene that encodes a citrate transporter, enhancing the Al-tolerance in rice by citrate secretion [78]. In our work were found several members of WRKY family in both inbred lines, but no one showed high similarity with previous WRKY described regulating the expression of genes under Al stress.
The zinc finger-associated to a homeodomain (ZF-HD) family regulates diverse plant-biological processes, such as development and responses to phytohormones and abiotic stresses [79]. An increase in the expression response of BraZF-HD under heat, cold, and salt stress was found in Chinese cabbage [79]. CONSTANS-like (CO-like) has been found to respond by the overexpression in ABA and salt stress as a positive regulator in Arabidopsis [80]. Q-Y Zeng, C-Y Yang, Q-B Ma, X-P Li, W-W Dong and H Nian [76] reported an important role for miR160 in the cleavage of Auxin Response Factor (ARF) transcripts in response to auxin, which might regulate the inhibition of root development under Al-stress. Little is known about the role of E2F/DP in abiotic stress but acts as a sub-category in response to heat stress in Arabidopsis and rice [81]. The relationships between ZF-HD, CO-like, and E2F/DP and aluminum stress are still poorly understood, although it is believed that this TFs might play diverse roles in transcriptional regulation of key genes in Al-stress response.
Some important Aluminium Resistance Transcription Factors that regulate the expression of genes related to Al tolerance have been identified in previous works. ART1 encodes a C2H2-type zinc finger TF required for Al resistance in rice [82], ART2 an ART1 homologs [83] and STOP1 that is involved in signal transduction pathways regulating aluminum responsive gene expression [84]. However, these TFs were not found in our analysis probably due the long term of exposure of Al toxicity levels. These TFs were induced in a short period of Al-exposure, regulating the expression of initial responsive genes in previous work. For these reasons, we believe that these TFs have been already expressed in initial steps in popcorn roots under Al-stress not being detected by the RNA-seq analysis.
Transposable elements (TE) are a source of spontaneous mutations [85][86][87] and play an important role in responding to environmental adverse conditions [88]. The beneficial mutations derivate by TEs may enhance plant adaptation to acid soils increasing the expression of Al-responsive genes or of the gene copy number [89]. On the other hand, TEs may reduce the expression of key genes affecting splicing patterns or altering protein function [89]. The TEs found in our data may contribute to activate or inactivate important genes that may confer tolerance to Al toxicity. However, further investigation has been made to characterize these TEs expressed under Al-stress. Moreover, up-regulated genes expressed only in Al-resistant lines might be useful as a marker to screening Al-resistance in maize, which might help identify key players in future studies. Transporters that presented positive and high fold change values, such as ABC transporter B family member 9 (Zm00001d044564 and Zm00001d043766) and member 19 (Zm00001d024600), ABC transporter G family 29 (Zm00001d036986), SWEET 2 (Zm00001d009365), SWEET 4A (Zm00001d015905), SWEET 13C (Zm00001d041067), SWEET 13A (Zm00001d023677), SWEET 14B (Zm00001d049252), Aluminum-activated malate transporter (Zm00001d046029), and aquaporin PIP2-2 (Zm00001d005410), must be involved in specific mechanisms of defense response against Al ion to our Al-resistant line. Once the transporters have an important role in Al-tolerance, these genes must be potential markers linked to Al tolerance candidate genes, and future studies should be performed to investigate the segregation of these genes.

Conclusions
We identified new players involved in Al-resistance response in popcorn using RNA-sequencing technology that were not found in previous studies using hydroponic experiments, thereby increasing our understanding of responses to Al-stress. Here, we found that the Al-resistant inbred line needs more than one effective mechanism to alleviate the damages caused by Al-stress and that the  [90,91]. Then, the treatment group was submitted to aluminum stress with 540 µM of AlCl 3 (160 μM Al 3+ ) at pH 4.5 for 72 h. The seedlings were maintained in a growth chamber at 25ºC with 12/12 h light/dark cycle. Total roots from three biological replicates were collected and immediately frozen in liquid nitrogen.

RNA isolation and transcriptome sequencing
The total root RNA was isolated with Trizol LS reagent (Invitrogen, USA) according to the manufacturer's protocol. The RNA was treated with DNase I Amp Grade (Thermo Scientific) to remove contaminated DNA, and then quantified by spectrophotometry (NanoDrop 2000c, Thermo Scientific).
The RNA integrity was verified by electrophoresis on 1.6% agarose gel in the presence of ethidium bromide. After quantification, the RNA samples were sent to Macrogen Inc. (Seoul, South Korea) where transcriptome sequencing library was generated using the TruSeq Stranded mRNA kit and sequenced using the Illumina HiSeq 2500 platform. Reads preprocessing and differential expression analysis Quality control was initially performed using the FastQC program (version 0.11.8) [92] to check the sequencing quality and identify reads with adaptors contamination. Then, the raw reads were

Real time qPCR
To validate the RNA-seq results, RNA from three independent replicates was treated with DNase I Amplification Grade (Invitrogen, USA) and the cDNA was synthetized from 2 µg of RNA using the SuperScript Reverse Transcriptase kit (Invitrogen, USA). Real-time qPCR for thirteen genes identified as differentially expressed at least one of the inbreed lines was performed with an ABI 7500 (Applied Biosystems, USA). The primers were designed using Primer Express software (Applied Biosystems, USA), and the specificity confirmed by BLAST in the Phytozome database (Additional file: Table S8).
The real-time qPCR reactions were performed using 1 μL of 1:10 diluted cDNA, 5 μL of primer Forward and Reverse mixed at 1.5 μM (each primer) and 6 μL of SYBR Green PCR Master Mix. The experiment was conducted using three biological replicates for each genotype and two technical replicates. The

Consent for publication: Not applicable
Availability of data and material: The data sets supporting the results of this article are available in the NCBI SRA repository, http://www.ncbi.nlm.nih.gov/bioproject/508768.

Competing interests:
The authors declare that they have no competing interests.  Tables   Table 1 Glutathione transferases proteins identified in RNA sequencing of Al-sensitive and Al-tolerant popcorn lines under 72 hours of Al-stress.   Table 3 Aluminum responsive transporters related in Al-resistant popcorn.    Gene ontology (GO) enrichment of biological process in Al-sensitive popcorn line.