Sugarcane partitions carbon into sucrose that can accumulate to 0.7 M in culms . This unique characteristic has been exploited and improved by humans through breeding. Studies that shed light on the molecular mechanisms behind this feature include gene expression and signaling studies on sink and source regulation , QTL studies for sucrose accumulation  and gene expression profiling during internode maturation [10, 11, 12, 38]. Such studies indicated that genes associated with sucrose metabolism are not abundantly expressed in culm tissues while genes related to synthesis and catalysis of sucrose are turned off during internode maturation. Genes involved in cellulose synthesis, cell wall metabolism and lignification are also regulated during this process. The activity of genes associated with internode development was evaluated in genotypes of S. robustum (which does not accumulate sucrose to high levels), S. officinarum and in a hybrid . Mature internodes of all three genotypes showed decreased expression of cell wall metabolism-associated genes and increased expression of genes related to sucrose metabolism. While the general conclusion of these studies does not appear to be in agreement, it is important to note that the genotypes, environment and age of plants used were different and that a larger sampling may be necessary to define gene profiles in sugarcane.
In this work, we evaluated mature and immature internodes of thirty genotypes using cDNA microrrays and qRT-PCR. Genes associated with sucrose content were defined through the analysis of segregating populations selected for one or three generations . Internodes 1, 5 and 9 (In1, 5 and 9) were collected from plants grown in the field. Among the genes found to be differentially expressed were those related to hormone signaling (auxin, ethylene, jasmonates), stress responses (drought, cold, oxidative), cell wall metabolism, calcium metabolism, protein kinases, protein phosphatases and transcription factors. We compared high Brix plants against low Brix plants by hybridizing pairwise the In1, In5 and In9 tissues directly (HB vs LB hybridizations) or by hybridizing mature against immature internodes (MI vs II). We validated gene expression by qRT-PCR in pools of clones and many individual genotypes. We also investigated if genes associated with sucrose content were responsive to sucrose or glucose treatments. Many of the sucrose-associated genes that are regulated during development are associated with drought responses or are modulated by ABA or sugars, as discussed below (see Additional file 2 and Table 1).
Protein kinases and calcium signaling
Protein phosphorylation appears to play a predominant role in sucrose accumulation and culm development. We have previously categorized sugarcane proteins with a PKinase domain using a phylogenetic approach and named sugarcane protein kinases (PKs) according to the groups obtained, similarity to other kinases and additional domains observed . We now add evidence that several of these genes are regulated during culm development.
A total of fifty-four genes corresponding to PKs, protein phosphatases (PPases) or receptor-like kinases (RLKs) were differentially expressed in high Brix plants or during culm maturation (see Additional file 2). ScMAPK-4 (SCSBAM1084E01.g) was more highly expressed in high Brix and mature internodes (Table 1). A MAPK kinase was reported to be involved in the regulation of source metabolism by glucose and stress, which is an indication that ScMAPK-4 might be important in establishing sink-source relationships in sugarcane [36, 41]. The most predominant PK category altered is the SNF1-like kinase family of proteins. In yeast, SNF1 regulates the expression of genes coding for carbohydrate metabolism and other metabolic enzymes . In plants, SNF1-related kinases have been named SnRK1  and comprise three distinct sub-families (SnRK1, SnRK2 and SnRK3). In sugarcane, we have identified members of all three sub-families . Analogous to SNF1, plant SnRK1s also regulate carbon metabolism at the level of gene expression. At least three important biosynthetic enzymes have been identified as biological substrates of SnRK1s: hydroxymethylglutaryl-CoA reductase (HMG-CoA reductase) ; sucrose-phosphate synthase  and nitrate reductase . It is possible to make a direct parallel between sucrose accumulation and the gene expression levels for an ScSnRK1 (SCJFRZ2032G01.g). ScSnRK1-2 and four 14-3-3 proteins of the GF14 type (SCCCLR1022D05.g, SCCCRZ1001D02.g, SCEQRT1031D02.g and SCEQRT1025D06.g) were expressed at lower levels in mature internodes (Table 1). 14-3-3 proteins, together with a SnRK1, phosphorylate and inhibit the enzyme sucrose phosphate synthase (SPS) in vitro [45, 47]. Our findings suggest that the decrease in the expression of these genes in the mature internodes may allow for increased sucrose accumulation. We also observed that ScSnRK1-2 was induced by sucrose treatment, while most of the ScCIPKs and ScPKABA and ScOSAPK genes were repressed (Table 1). This is an interesting finding that may functionally distinguish the pathways triggered by these kinases in response to sucrose and stress.
Members of the SnRK2 and SnRK3 sub-family including two Osmotic Stress-Activated Kinases – OSA-PK (SCEPRZ1009C10.g and SCCCST1004A07.g) and three CBL-interacting Protein Kinases – CIPK (SCJFRZ2032C08.g, SCMCRT2103B04.g and SCJLRZ1023H04.g) were identified as developmentally regulated during culm maturation (Table 1). Most importantly, three CIPKs (SCBFSB1046D04.g, SCMCRT2103B04.g, SCCCLR1C05B07.g) were more highly expressed in high Brix plants. CBL are regulatory subunits similar to calcineurin that bind to and respond to calcium signals . It has been shown that OSA-PKs and CIPKs mediate drought, osmotic, saline and cold stresses in response to ABA and calcium . Among our differentially expressed genes we found nine genes associated with calcium signaling (SCVPLR1049C09.g, SCCCRZ1C01H06.g, SCJLLR1108H07.g, SCEZLB1012F10.g, SCCCAM1001A03.g, SCAGLR1043F02.g, SCCCCL3120G07.g, SCCCRZ1003A03.g, SCRFLR2037F09.g) and a calcium-dependent protein kinase (SCEQRT2099H01.g – ScCDPK-27) that also indicates a role for this second messenger in sucrose accumulation in sugarcane (Table 1). Sucrose synthesis control depends on the activity of the sucrose phosphate synthase, which catalyses the synthesis of sucrose 6-phosphate from UDP-glucose and fructose 6-phosphate. Sucrose breakdown depends on the activity of invertase, which breaks down sucrose into glucose and fructose, and on the activity of sucrose synthase, that converts sucrose in fructose and UDP-glucose in the presence of UDP . Several studies have shown that some CDPKs phosphorylate and regulate sucrose synthase [50–53]. Studies on the maize sucrose synthase showed that phosphorylation of this enzyme on the Ser-15 by CDPKs stimulates the sucrose breakdown activity of this enzyme [50, 52]. Besides, CDPKs can phosphorylate residue Ser-170 of this enzyme directing it to the degradation pathway via proteosome 26S [52, 54]. The decrease in expression of ScCDPK-27 in the mature internode correlates well with increased sucrose in this organ. The activity of sucrose synthase modulates the source-drain relationship [55, 56], which eventually determines sucrose content in sugarcane internodes. Additionally, some CDPKs can phosphorylate and inactivate the enzyme sucrose phosphate synthase [57, 58], which might contribute to lower sucrose in culms when this enzyme is expressed in high levels, such as seen in low Brix genotypes. Since sucrose biosynthesis is a process regulated by calcium, CDPKs and SnRKs, the genes differentially expressed observed in the high Brix genotypes may all contribute and act as critical control points in sucrose accumulation in this grass.
We found a prevalence of gene families regulated by ABA, drought and other stresses among the genes associated with sucrose content . Sixty-nine genes associated with sucrose content were identified to be regulated in response to drought and eleven to ABA (see Additional file 2). This is a strong indication that some of the pathways associated with sucrose content and culm development may overlap with stress signaling pathways. A similar finding was described by Casu and colleagues that found many stress-related genes expressed in culms . Overall, around 32% of the genes previously found to be responsive to drought are associated with sucrose content (Figure 1). It is generally known that sugarcane plants maturation is favoured by the exposure to a period of low water precipitation. It is possible that increased sucrose content is dependent on a drought season and that plants converge the drought and sucrose signaling pathways to sustain growth even during the stress season. Sugars that transduce stress signals or act as osmoprotectants, like fructose, raffinose and trehalose  could be central players during this process. A PP from the PP2C family (SCEPRZ1010E06.g) similar to a PPase that transduces the ABA signal was identified as associated with Brix, drought and ABA (Table 1). This PPase was less expressed in high Brix, reduced in the mature internodes and induced by drought and ABA. A similarity search showed that this PP2C is very similar to ABI1 and ABI2 from Arabidopsis thaliana. PP2Cs that are part of the ABA signaling pathway, such as ABI1, ABI2, AtPP2CA and AtP2CHA, have their transcript levels increased by this phytohormone [59, 60]. Among the processes regulated by ABI1 and ABI2 is stomatal closure, which is certainly one of the first protective measures during drought signaling. Moreover, some of the ABA biosynthesis enzymes are induced by drought and osmotic stress . The fact that sugarcane genes associated with ABA and drought signaling are differentially regulated in plants with distinct sucrose accumulation capacities is an indicative that the role of ABA is well worth being further verified.
Drought responses vary depending on the duration and intensity of the stimulus and comprehend protective alterations and long term growth changes . Many genes responsive to drought have been catalogued [14–18]. The drought stimulus lead to transient calcium fluxes, the activation of calcium sensors, the accumulation of reactive oxygen species, the activation of the MAPK pathway and the induction of several transcription factors including DREB2A, DREB2B  and NAC . We have found, among the genes associated with sucrose content, many components of the gene families mentioned above. A MAPK was more expressed in high Brix and in mature internodes and repressed by ABA (SCSBAM1084E01.g), a DREB was induced during culm maturation (SCCCLR1001D10.g) (Table 1) and over forty stress responsive genes were identified (Additional file 2). A DEHYDRIN TYPE 1 (SCQGLR1085F11.g) regulated by the DREB signaling cascade  was dramatically repressed in mature internodes of high and low Brix plants and induced after 72 h and 120 h drought treatment (Table 1). A barley dehydrin gene, DHN1, was highly expressed in cells cultured at 25°C and 2°C in media containing high sucrose but our data indicated the dehydrin expression may not be a consequence to high sucrose since immature internodes do not have high levels of this sugar . Overexpression of DREB2A in Arabidopsis thaliana led to the generation of transgenic plants more tolerant to drought [66, 67]. It is possible that some of the genotypes may indeed be more resistant to drought and thus able to grow and accumulate more sucrose, but additional experiments are necessary to verify this hypothesis.
Transcription factors and hormone signaling
We have recently integrated and evaluated the SUCEST and PlantGDB http://planttfdb.cbi.pku.edu.cn/web/index.php?sp=so EST databases for putative Transcription Factors and identified 2,406 candidate TFs. These were classified into families and can be found at http://grassius.org. We found twenty-one transcription factors (TFs) regulated during culm development (Table 1). The great majority was more expressed in the immature internodes, including two members of the Homeobox Knotted1-homeodomain (SCAGLR1021G10.g and SCRLAM1010D08.g), which have been shown to be involved in developmental processes in maize . Developmentally regulated genes include a homolog (SCBFAD1046D01.g) to anthocyanin regulatory R-S protein containing a helix-loop-helix (HLH) domain, that controls tissue-specific synthesis of anthocyanin pigments . Nine transcription factors were identified as differentially expressed when high Brix and low Brix genotypes were compared including an ARF6 (AUXIN RESPONSE FACTOR6a) (SCEZLB1010E10.g), a NAM (NO APICAL MERISTEM) (SCCCLR2003E10.g) and an EIL (ETHYLENE INSENSITIVE3-LIKE) (SCCCRZ1004H12.g) (Table 1). The NAM transcript was less abundant in both populations analysed, negatively regulated by sucrose and glucose treatment and induced by drought (Table 1 and Figure 4). NAM transcription factors in Oryza sativa have been described as important regulators of drought tolerance . This may indicate a connection between these signaling pathways , possible co-regulation associated with sucrose content and cross-talks or signaling overlaps between sugar sensing, sugar mobilization and drought responses.
Among the genes more expressed in the immature internodes, we also found several genes similar to auxin, ethylene and giberellin-responsive TFs (Table 1). We found a second TF related to ARF6 (SCCCLR1024F10.g) and four AUXIN RESPONSE PROTEINS (SCCCRZ1001G10.g, SCVPLR2005H03.g, SCJFRZ2009F04.g, SCJLLR1054C09.g) more expressed in immature internodes. Signaling by auxins during culm development was also pointed out by the altered expression of two nitrilase genes (SCEQRT1028H06.g and SCRFLR1012D12.g) with a putative role in this hormone's biosynthesis, an auxin efflux carrier (SCCCAM2004G02.g) and a AUXIN REPRESSED PROTEIN (SCCCLR2002F08.g) which is up-regulated in mature internodes. One of the nitrilases (SCEQRT1028H06.g) was induced after 72h of drought  and its expression level was confirmed by qRT-PCR (Figure 4). Another drought-induced nitrilase (SCCCCL6002B05.g) was found more expressed in high Brix plants. This gene is highly similar to the maize ZmNIT2 gene, which converts indole-3-acetonitrile to indole-3-acetic acid . Overall, differential expression of auxin signaling genes during internode development and/or association with sucrose content was observed in twenty different biological samples.
Ethylene was observed as a putative modulator of this process (Table 1). One EIL was found less expressed in high Brix plants (SCCCRZ1004H12.g) and two were less expressed in mature internodes (SCBGFL4052C11.g and SCCCRZ1004H12.g). Besides, one ACC oxidase (SCVPLR2012A10.g) was found to be more expressed in high Brix plants and less expressed in mature internodes.
Jasmonic Acid biosynthesis also seems to have a relevant role in culm development since several enzymes envolved in methyl jasmonate biosynthesis were found to be more expressed in immature internodes, two lipoxygenases (SCCCRT1001E01.g and SCJFRT1007H07.g) and an Omega-6 fatty acid desaturase (SCCCLR1C03G01.g) (Table 1).
Cell wall biosynthesis
There were several genes with a putative function in cell wall metabolism that were among the differentially expressed genes, such as the expansins SCQGRT1040G03.g and SCCCLR2C02A05.g (Table 1). Similar genes were found to be expressed in two-day-old rice seedlings, a stage where rapid cell elongation occurs accompanied by cell division . The authors believe EXP activity may be required for cell expansion. Expansins may act in the relaxation of the cell wall, possibly by breaking the bonds between cellulose microfibrils and matrix polysaccharides [74, 75] allowing for cell expansion. Our data indicates that, in sugarcane, a gene similar to EXPA23 (SCQGRT1040G03.g) is more expressed in 7-month old high Brix plants as compared to low-Brix plants, and the EXPA11 (SCCCLR2C02A05.g) in turn, is more expressed in low Brix plants (after 11 months of planting) (Table 1). As mentioned above, we have evidence that auxin signaling is highly active in immature internodes. Auxin signaling is associated with plant cell expansion , which may be an additional evidence that the high Brix plants selected have cell expansion alterations that might confer higher sucrose accumulation capacity. These observations are corroborated by the identification of a XYLOGLUCAN ENDO-B-1,4 GLUCANASE (XTH) (SCBFLR1039B05.g) that is more expressed in immature internodes (Table 1). XTHs can hydrolyse xyloglucans, major components of plant cell walls, and transglycosylate residues into growing xyloglucan chains, that may be important during tissue expansion . Our data is in agreement with previous findings from Casu and colleagues  that identified five XTHs and four β-expansins less expressed in mature internodes, as well as two caffeoyl-CoA O-methyltransferases induced in mature internodes.
We found five genes of the lignin biosynthesis pathway associated with sucrose content (Table 1). The first step in lignin biosynthesis in plants is the deamination of L-phenylalanine by Phenylalanine Ammonia-Lyase (PAL) to cinnamic Acid. PAL is the first enzyme of the phenylpropanoid pathway . It converts L-phenylalanine into trans-cinnamic acid (t-CA), which is further transformed in plants into many phenylpropanoid compounds, such as lignins, antioxidants, anthocyanins and flavonoid nodulation factors. We found three PAL genes associated with sucrose content and more expressed in immature internodes (SCCCLR1048D07.g, SCEQRT1024E12.g and SCSGAM1094D05.g). SCEQRT1024E12.g was induced after ABA treatment and repressed after 72 and 120 h drought stress. The second step in lignin biosynthesis is catalyzed by a cinnamate 4-hydroxylase (C4H) . We found one SAS less expressed in high Brix similar to a C4H (SCSGFL4193B05.g). Down the pathway p-Coumaroyl is transformed into Caffeoyl CoA by a p-coumaroyl shikimate 3'-hydroxylase (C3H). One SAS similar to a C3H (SCACSB1037A07.g) was found less expressed in high Brix plants. We also observed a FERULATE 5-HYDROXYLASE (F5H) (SCEZHR1087F06.g) and a CAFFEIC ACID 3-O-METHYLTRANSFERASE (COMT) (SCRFLR1012F12.g) more expressed in mature internodes. F5H was less expressed in high Brix while COMT was induced (Table 1). While all the above mentioned genes may have a role in cell wall metabolism, it is important to note that trans-cinnamic acid can also be converted into salicylic acid and anthocyanins  and, until the activity of these enzymes is verified, the data can only indicate a putative alteration in cell wall biosynthesis and modification in the accumulation of sucrose in culms.
Cell wall biosynthesis can reduce sucrose accumulation since carbon fluxes directed to plant growth and cell wall expansion may alter carbon partitioning into sucrose. It is also possible that sucrose accumulation may trigger increased lignification. One of the PAL enzymes was induced by sucrose treatment up to 14-fold indicating that this enzyme is highly responsive to sucrose. An induction of a COMT gene has already been described during culm maturation  but this is the first report implicating a PAL, C4H, C3H, F5H and COMT in sucrose content. It is possible that some of the genotypes analyzed also differ in biomass content and a continued agronomic evaluation is necessary to assess how gene expression in the selected genotypes is related to other characteristics, such as cell wall composition, growth rates, internode size and width, number of internodes and drought tolerance, for instance. Many parameters besides Brix may differ among the genotypes and have not yet been evaluated. It is possible also that the high Brix genotypes may be more amenable for acid and enzymatic hydrolysis and cellulosic ethanol production. Silencing of lignin biosynthesis genes has been shown to benefit sugar release for lignocellulosic biomass fermentation ; it will be thus interesting to test if altered biomass has been selected for during the breeding process. At any rate, the alteration of these cell wall biosynthesis genes in association to Brix content is an interesting indication of a correlation between these processes.
A relationship between high sugar content and sugar signaling
We showed that a set of genes associated with sucrose content is also early sugar-responsive. Since most of these genes are related to signal transduction (kinases, phosphatases, transcription factors, hormone synthesis) they are likely to constitute upstream components of the sugar regulatory cascade. These findings raise the interesting possibility that sugar signaling may somehow influence sugar accumulation capacity in sugarcane. How these genes may influence sucrose accumulation is an open question. Interestingly, between sucrose/glucose-treated young seedlings and high Brix genotypes, contrasting expression patterns were found for 15 genes, while the remaining 9 genes presented similar regulatory trends (Table 1 and data not shown). This latter set of genes may be related to higher sugar fluxes and/or higher sugar sensitivity in high Brix genotypes. Opposite regulatory patterns between young seedlings and internodes of high Brix genotypes are more difficult to explain, but could reflect differential developmental-dependent controls. A comparative analysis with Arabidopsis showed that among the twenty-four sugarcane genes tested, five Arabidopsis-sugarcane probable groups of orthologues and two pairs of Arabidopsis-sugarcane close homologues (sister clades) were apparently regulated similarly by sugars in seedlings (see Additional file 4). Orthologous genes of the signal transduction-class, such as those encoding transcription factors or kinases with conserved regulatory features, are likely to represent important players in the sugar signal transduction pathways and this can now be tested. Within this framework, it should also be interesting to further analyze the integration/interaction of the Arabidopsis CUC1/NAC-type transcription factor (At3g1550), which controls shoot apical meristem formation  in the sugar regulatory network.
Data validation across genotypes
To confirm gene expression and evaluate transcript levels we performed qRT-PCR reactions for forty-two genes. With a probability value higher than 0.95 we observed that 80% of the gene expression data obtained using cDNA microarrays were compatible with the qRT-PCR data.
Validation of developmental regulation was elucidative of differences among high Brix and low Brix populations. ScCIPK-21 for instance, a gene more expressed in high Brix and in mature internodes, was found to be much more induced during culm development in the high Brix plants, what may be an indicative that induction of this gene may lead to higher sucrose levels. A category that was consistently more expressed in immature internodes and high Brix and that has been seen to be responsive to drought in other plants is the aquaporin family of proteins. We wanted to verify if genes of this family could be useful expression markers of sucrose content. Five aquaporins, from both the MIP and PIP sub-families (SCCCRZ1002E08.g, SCCCST3001H12.g, SCEQRT2100B02.g, SCCCLR1024C03.g, and SCCCRZ1001F02.g) were regulated during culm development and two of them found to be associated with high Brix (SCCCST3001H12.g and SCEQRT2100B02.g) in population 2 (Table 1). This large family of proteins is primarily involved in the regulation of water movement between cells and cell compartments, although many of them also facilitate the passage of small solutes [83, 84]. The accumulation of sucrose in such high concentrations as seen in sugarcane cells certainly represents an osmotic challenge that demands efficient control of solute compartmentalization and water transport out of the vacuoles. As key players in the equilibration of water potentials via regulation of membrane permeability, aquaporins may have a fundamental role in the process of sugar storage in sugarcane vacuoles. It has been observed in Arabidopsis that loss of the aquaporin TIP1.1 severely affects carbohydrate metabolism and transport , and the authors postulate that this aquaporin could be involved in a vesicle-based routing of carbohydrates towards the central vacuole. In our study, expression of one aquaporin correlated to lower sucrose content in both populations analyzed. Since differential expression was determined in pools of seven or eight individuals we decided to verify how many of the genotypes in those pools presented the observed expression patterns. Expression data was obtained using qRT-PCR for twelve of the sixteen extreme individuals of Population 1 (Figure 5). We calculated the average expression levels across all twelve genotypes for the Aquaporin gene (SCCCRZ1002E08.g) and observed that the gene was less expressed in all high Brix genotypes and more expressed in half of the low Brix genotypes. This is a strong indicative that low expression of this gene has been segregated and selected by the breeding process and is strongly associated with high sucrose content. To verify if this would be the case for other differentially expressed genes we evaluated the expression profiles of eleven genes in the twelve extreme genotypes of Population 1. For some genes, such as the HLH TF (SCCCRZ1001H05.g), expression was consistently lower than average in all low Brix genotypes, but found to be higher in only two of the high Brix genotypes (Figure 5). In this case, the two genotypes had almost 10,000 fold increased expression for the genes, which may account for the differential expression observed in the pool used for cDNA microarray analysis. This is an extreme case and, in general, most of the data shows around 50% of validation in the individual genotype validations.