Sugarcane genes associated with sucrose content

Abstract

Background: Sucrose content is a highly desirable trait in sugarcane as the worldwide demand for cost-effective biofuels surges. Sugarcane cultivars differ in their capacity to accumulate sucrose and breeding programs routinely perform crosses to identify genotypes able to produce more sucrose. Sucrose content in the mature internodes reach around 20% of the culms dry weight. Genotypes in the populations reflect their genetic program and may display contrasting growth, development, and physiology, all of which affect carbohydrate metabolism. Few studies have profiled gene expression related to sugarcane's sugar content. The identification of signal transduction components and transcription factors that might regulate sugar accumulation is highly desirable if we are to improve this characteristic of sugarcane plants.

Results: We have evaluated thirty genotypes that have different Brix (sugar) levels and identified genes differentially expressed in internodes using cDNA microarrays. These genes were compared to existing gene expression data for sugarcane plants subjected to diverse stress and hormone treatments. The comparisons revealed a strong overlap between the drought and sucrose-content datasets and a limited overlap with ABA signaling. Genes associated with sucrose content were extensively validated by qRT-PCR, which highlighted several protein kinases and transcription factors that are likely to be regulators of sucrose accumulation. The data also indicate that aquaporins, as well as lignin biosynthesis and cell wall metabolism genes, are strongly related to sucrose accumulation. Moreover, sucrose-associated genes were shown to be directly responsive to short term sucrose stimuli, confirming their role in sugar-related pathways.

Conclusion: Gene expression analysis of sugarcane populations contrasting for sucrose content indicated a possible overlap with drought and cell wall metabolism processes and suggested signaling and transcriptional regulators to be used as molecular markers in breeding programs. Transgenic research is necessary to further clarify the role of the genes and define targets useful for sugarcane improvement programs based on transgenic plants.

Figure 1

Comparison of differential gene expression associated with sucrose content, culm development and drought responses in sugarcane. Genes were identified as associated with sucrose content if they were differentially expressed when high Brix or low Brix pools of plants were compared. Genes regulated during culm development were identified by comparing Mature and Immature Internodes. The drought-responsive genes were found to be induced or repressed by drought after 24, 72 or 120 h of water deficit. The figure represents a Venn diagram of the three differential expression data sets. Technical replicates range from 2 to 16 since genes are spotted several times in the same array. The credibility level used to define outliers was 0.96 in all three data sets.

Figure 2

Real Time PCR (qRT-PCR) analysis of Populations gene expression. The y axis refers to the relative expression ratio between target mRNA versus the reference mRNA (polyubiquitin-PUB SCCCST2001G02.g). The relative expression levels were determined in Internode 1, 5 and 9 tissues from a pool of the eight individuals with the highest Brix measures (HB) and the eight individuals with the lowest Brix measures (LB) from Population 1 (A) and from a pool of the seven individuals with the highest Brix measures (HB) and the seven individuals with the lowest Brix measures (LB) from Population 2 (B). The reactions for the target mRNA and reference mRNA were carried out in parallel and each reaction was performed in triplicates. Error bars were calculated as described previously [31]. The transcript levels for the reference genes were verified not to vary in response to the treatments. The values of P correspond to the probability Pr(HB>LB) and Pr(HB<LB) for up- and down-regulated genes, respectively. The SAS was considered differentially expressed when P ≥ 0.95.

Figure 3

Real Time PCR (qRT-PCR) analysis of internode developmental gene expression. The y axis refers to the relative expression ratio between target mRNA versus the reference mRNA (polyubiquitin SCCCST2001G02.g). The relative expression levels were determined in Internode 1 and 9 tissues from a pool of the seven individuals with the highest Brix measures (HB) and the seven individuals with the lowest Brix measures (LB) of Population 2. All reactions were carried out in parallel and each reaction was performed in triplicates. Error bars were calculated as described previously [31]. The transcript levels for the reference genes were verified to not vary in response to the treatments. The P values correspond to the probability Pr(MI>II) and Pr(MI<II) for up- and down-regulated genes, respectively when In9 and In1 samples were compared. The values of P were calculated for the HB and LB pools of plants independently. The SAS was considered differentially expressed when P ≥ 0.95.

Figure 4

Real Time PCR (qRT-PCR) analysis of drought and ABA-responsive gene expression. The y axis refers to the relative expression ratio between target mRNA versus the reference mRNA (polyubiquitin SCCCST2001G02.g; GAPDH Gene ID: 542367; UBE2 SCBGLR1002D06.g) in sugarcane plants treated with ABA for 12 h or drought conditions for 24, 72 or 120 h. The reactions for the target mRNA and reference mRNA were carried out in parallel and each reaction was performed in triplicates. Error bars were calculated as described previously [31]. The transcript levels for the reference genes were verified to not vary in response to the treatments. The values of P correspond to the probability Pr (Treated>Control) and Pr (Treated<Control) for up- and down-regulated genes, respectively. The SAS was considered differentially expressed when P ≥ 0.95.

Figure 5

Real Time PCR (qRT-PCR) analysis of individual genotypes gene expression. The y axis refers to the relative expression ratio between target mRNA versus the reference mRNA (polyubiquitin SCCCST2001G02.g). The relative expression levels were determined in Internode 1, 5 and 9 tissues from six individuals with the highest Brix measures (CTC98-241, CTC98-242, CTC98-243, CTC98-244, CTC98-246 and CTC98-253) and six individuals with the lowest Brix measures (CTC98-261, CTC98-262, CTC98-265, CTC98-272, CTC98-277 and CTC98-279) of Population 2. All reactions were carried out in parallel and each reaction was performed in triplicates. Error bars were calculated as described previously [31]. The transcript levels for the reference genes were verified to not vary in response to the treatments. The significance of differential gene expression was determined considering normal distributions for each tested condition and comparing them to the average expression for all samples (dotted line). The values of P correspond to the probability Pr (GenotypeX>average) and Pr (GenotypeX<average) for up- and down-regulated genes. P values were calculated for each genotype independently. The SAS was considered differentially expressed when P ≥ 0.95.

Figure 6

Quantitative PCR (qRT-PCR) analysis of sucrose and glucose responsive genes. The y axis refers to the relative expression ratio between target mRNA versus the reference mRNA (tubulin SCCCRZ1002H03.g) for 3 different experiments in sugarcane thirteen-old day seedlings treated with 3% glucose and 3% sucrose for 4 h. R1, R2 and R3 refers to three control and three sucrose and glucose independent treatments. Error bars were calculated as described previously [31]. The transcript levels for the reference genes were verified to not vary in response to the treatments. The values of P correspond to the probability Pr (Treated>Control) and Pr (Treated<Control) for up- and down-regulated genes, respectively. The SAS was considered differentially expressed when P ≥ 0.95.