**5. Transcriptomic analysis of an elite Mexican sugarcane cultivar ('Mex 69-290') in response to osmotic stress. Identification of genes with biotechnological potential**

Modern sugarcane cultivars have been obtained by inter-specific hybridizations between the high-sucrose-yielding of *S. officinarum* (2n = 8x = 80) and the stress-tolerant *S. spontaneum* (2n = 40–128). As a consequence, sugarcane cultivars present large (10 Gb) and poly-aneuploid genomes with numerous gene alleles and repetitive sequences. Such genome complexity has made it difficult to obtain a complete sequenced reference genome that could aid in the identification of novel genes with biotechnological potential for the improvement of this important C4 crop. Alternatively, de novo transcriptome assembly of reads produced by highthroughput sequencing technologies (also referred to as Next Generation Sequencing (NGS)) offers a mean to unravel global gene expression changes in response to various conditions in sugarcane. For example, some recent works have employed High-throughput sequencing to identify sugarcane genes involved in leaf abscission [49], biomass content and composition [50], and abiotic stress [51]. Li and cols. [49] performed a transcriptome analysis to identify genes associated with leaf abscission in sugarcane. They employed the Illumina HiSeq 2000 platform (2x90pb) to analyze six cDNA libraries from parents and their F1 offspring, which present different leaf abscission behaviors. After a total assembly, they found 275,018 transcripts corresponding to 164,803 genes. Then, to identify genes related to leaf abscission in sugarcane [49], analyzed a core set of 1, 202 transcripts which were up-regulated in leaf abscission sugarcane plants (LASP) in comparison to leaf packaging sugarcane plants (LPSP). They found that some of these genes were associated with plant-pathogen interaction, response to stress, and ABA-associated pathways. On the other hand [50], performed an extensive transcriptome analysis to identify genes associated with biomass content. They employed the Illumina HiSeq 4000 platform to analyze cDNA libraries from 20 internodal samples of 10 different sugarcane genotypes, which were divided in low and high fiber containing groups. They found 5601 and 4659 unique expressed transcripts in High and Low fiber containing genotypes; and 83,421 shared expressed transcripts between both groups. Furthermore, they found 555 differentially expressed transcripts between low and high fiber containing genotypes. Of these, 151 and 23 transcripts corresponded to sugar and fiber accumulation, respectively. Some of these genes were involved in Carbohydrate metabolism, Photosynthesis, Cell-wall metabolism and Lignin Pathway; DIR proteins were also represented [50].

The best results out of the measured parameters were obtained from the continuous immersion propagation system. It was concluded the reason for this may reside in the elimination of gelling agent, which additionally lowers production costs in the process of delivering this sugarcane's variety to the field. Plants obtained under this system achieved normal development, they developed shoots and roots cyclically and no vitrification was detected in any of the evaluated micropropagation phases. This suggests that the clone obtained from the MEX69290 variety is tolerant to liquid culture conditions. Apparently this system does not generate an abiotic stress, stationing it as a prospective medium to perform genetic transformation processes and to study its gene expression pattern that could further make enhanced

**Figure 4.** Greenhouse adaptation of *in vitro* plants of *S. officinarum* var. MEX69290, from culture: (A) semi-solid; (B) continuous immersion; (C) temporary immersion. Substrate consisted on a 3: 1 mixture of sunshine: soil. All plantlets

**5. Transcriptomic analysis of an elite Mexican sugarcane cultivar ('Mex 69-290') in response to osmotic stress. Identification of genes** 

Modern sugarcane cultivars have been obtained by inter-specific hybridizations between the high-sucrose-yielding of *S. officinarum* (2n = 8x = 80) and the stress-tolerant *S. spontaneum* (2n = 40–128). As a consequence, sugarcane cultivars present large (10 Gb) and poly-aneuploid genomes with numerous gene alleles and repetitive sequences. Such genome complexity has made it difficult to obtain a complete sequenced reference genome that could aid in the identification of novel genes with biotechnological potential for the improvement of this important C4 crop. Alternatively, de novo transcriptome assembly of reads produced by highthroughput sequencing technologies (also referred to as Next Generation Sequencing (NGS)) offers a mean to unravel global gene expression changes in response to various conditions in

tolerant clones.

**with biotechnological potential**

survived 100% after 30 days in the greenhouse.

96 Plant, Abiotic Stress and Responses to Climate Change

Regarding abiotic stress, Belesini and cols. [51] analyzed the transcriptomic profile of the drought-tolerant 'SP81-3250' and the drought-sensitive 'RB855453' sugarcane cultivars under drought stress conditions for 30, 60, and 90 days. They analyzed a total of 54 cDNA libraries by Illumina HiScanSQ System and HiSeq 2500 platforms. Among the genes that were induced in the drought-tolerant cultivar, they found an ascorbate peroxidase, a MYB TF, an E3 SUMOprotein ligase SIZ2, a coenzyme A ligase (a key enzyme for the biosynthesis of flavonoids), and an aquaporin, among others. These types of genes are well known to play a role in abiotic stress tolerance. In the drought-sensitive cultivar they found several kinases that were induced upon stress like Receptor like protein kinases (RLK), which might play a role in stress stimulus perception; bHLH transcription factors; ACC oxidase from the ethylene biosynthetic pathway; and many undescribed genes. More recently (2017), in our laboratory Pereira-Santana and cols. [52] analyzed the transcriptomic profile of the 2nd most important sugarcane cultivar in Mexico, 'Mex 69-290', in response to osmotic stress. In such study, authors employed the High-throughput sequencing system HiSeq-Illumina (2x100bp) to analyze 16 cDNA libraries representing leaves and roots of *in vitro*-grown plantlets exposed to PEG-8000 during 0, 24, 48, and 72 hours. After assembly of a total of 140, 339 unigenes, Pereira-Santana and cols. Found core sets of 536 and 750 up-regulated genes in response to osmotic stress in roots and leaves, respectively; and core sets of 1093 and 531 down-regulated genes in roots and leaves, respectively. After gene annotation, the authors found that sugarcane 'MEX69290' responds to osmotic stress by increasing the expression of genes involved in transcription regulation, oxide-reduction, carbohydrate catabolism, and flavonoid and other secondary metabolites biosynthesis. Genes responsive to ABA, water deprivation, and heat stress were also up-regulated. On the other hand, this sugarcane cultivar responds to osmotic stress by decreasing the expression of genes involved in sucrose and starch metabolic processes, cell wall biogenesis, cellulose biosynthesis, anion transport, and light response. A handful of the genes found by Pereria-Santana and cols. Are presented along with their expression profiles in the heat map of **Figure 5A**. Because of the well-defined expression pattern of some of these genes, they could prove to be useful as expression markers in the response of 'MEX69290' to osmotic stress. For example, ABA 8-hydroxylase 3, Isoflavone 2-hydroxylase, LEA 14A, and NAC TF 25 showed clear patterns of up-regulation. In fact, in our laboratory further expression and functional analyses are currently being carried out regarding this NAC TF25 gene. Conversely, Bidirectional sugar transporter SWEET11, Cellulose synthase E6, and Sugar transporter ERD6 16 showed clear patterns of down-regulation. These down-regulated genes are also interesting, not just because of their responsiveness to osmotic stress but also due to their involvement in sucrose metabolism. The engineering of these genes might increase biomass production in sugarcane and tolerance to osmotic stress simultaneously. Furthermore, many TFs known to play important roles in the stress responses of plants, i.e. HSF, ZN, bZIP, WRKY, NAC, and MYB, were found in abundance in the total assembly of the 'MEX69290' transcriptome (**Figure 5B**). Even when some of these TF families seemed underrepresented (like NAC and MYC), they still provide a useful benchmark to conduct phylogenetic, expression, and functional analysis.

In addition to the insights about the global gene expression dynamics of 'Mex 69-290' in response to osmotic stress and the identification of novel TFs, the work of Pereira-Santana and cols. Provides a useful benchmark for the study of other specific gene families of biotechnological significance for sugarcane engineering, for example the DIR protein family. Plant DIR proteins are believed to be involved in lignin biosynthesis, defense [56, 57], and abiotic stress responses such as dehydration [58], and salinity and oxidative stress [59]. In a recent study, 5 available sequence databases for sugarcane were surveyed, a total of 120 DIR proteins were identified [60]. Phylogenetic analysis showed that these DIR proteins are divided in 64 groups and 7 major clades: Dir-a, Dir-b/d, Dir-c, Dir-e, Dir-g, Dir-h, and Dir-i [60]. In the sugarcane transcriptome assembly of 'sugarcane Mex 69-290' performed in our laboratory by Pereira-Santana and cols, a total of 48 predicted proteins with DIR-like domains were identified. These DIR proteins were clustered in 7 groups according to their expression patterns (**Figure 6**). DIR42 protein from cluster 1 was significantly up-regulated in all time points of osmotic stress in root tissues. Conversely, DIR40 protein from cluster 7 was significantly down-regulated in all time points of osmotic stress in leaf tissues. In general, DIR genes from cluster 4 seem to possess a relative high expression in roots under control conditions, and those from cluster 7 seem to possess a relative high expression in leaves under control conditions. DIR genes from both clusters are down-regulated in response to osmotic stress. On the other hand, we also recovered a homolog of the ScDir gene (GenBank: JQ622282.1) from the sugarcane variety FN39 (DIR38 in cluster 5). The expression of ScDir from FN39 has been

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**Figure 6.** Differential expression in response to osmotic stress of 48 Dirigent proteins found in sugarcane 'MEX69290' transcriptome. The 48 Dirigent sequences from sugarcane were grouped according to their expression profiles in 7 clusters (1–7). Data was obtained from Pereira-Santana and cols [52]. Heat map and sequence clustering were generated with ComplexHeatmap v1.14.0 [53] in R v3.4.1 [54] using the "euclidean" distance method and "complete" clustering

method.

**Figure 5.** Selected DEGs in response to osmotic stress and abundance of major TF families and Dirigent protein family in sugarcane 'MEX69290' transcriptome. (A) Expression profile of 20 selected DEGs in leaves and roots of sugarcane 'MEX69290' plantlets submitted to PEG-8000 treatment during 0, 24, 48, and 72 hours. Data was obtained from the work of Pereira-Santana and cols [52]. The heat map was generated with the ComplexHeatmappackage v1.14.0 [52] in R v3.4.1 [53]. (B) Abundance of major stress-related TF families and Dirigent protein family in arabidopsis, rice, sorghum, and sugarcane. The results were obtained by means of HMM searches using the profiles of the HSF (PF00447), ZF (PF00096), bZIP (PF00170), WRKY (PF03106), NAC (PF02365), MYB (PF00249), and Dirigent (PF03018) proteins obtained from the Pfam database (http://pfam.xfam.org) [54]. For this analysis the complete predicted proteomes (primary transcripts only) of arabidopsis, rice, and sorghum were obtained from Phytozome v. 12 [55]. Sugarcane predicted protein dataset was obtained from the transcriptome assembly of Pereira-Santana and cols [52] HMM searches were performed using HMMER3 v3.1b2 (http://hmmer.org/) and set to a cut-off e-value of 1e-05 and a score above the inclusion threshold of each HMM profile.

In addition to the insights about the global gene expression dynamics of 'Mex 69-290' in response to osmotic stress and the identification of novel TFs, the work of Pereira-Santana and cols. Provides a useful benchmark for the study of other specific gene families of biotechnological significance for sugarcane engineering, for example the DIR protein family. Plant DIR proteins are believed to be involved in lignin biosynthesis, defense [56, 57], and abiotic stress responses such as dehydration [58], and salinity and oxidative stress [59]. In a recent study, 5 available sequence databases for sugarcane were surveyed, a total of 120 DIR proteins were identified [60]. Phylogenetic analysis showed that these DIR proteins are divided in 64 groups and 7 major clades: Dir-a, Dir-b/d, Dir-c, Dir-e, Dir-g, Dir-h, and Dir-i [60]. In the sugarcane transcriptome assembly of 'sugarcane Mex 69-290' performed in our laboratory by Pereira-Santana and cols, a total of 48 predicted proteins with DIR-like domains were identified. These DIR proteins were clustered in 7 groups according to their expression patterns (**Figure 6**). DIR42 protein from cluster 1 was significantly up-regulated in all time points of osmotic stress in root tissues. Conversely, DIR40 protein from cluster 7 was significantly down-regulated in all time points of osmotic stress in leaf tissues. In general, DIR genes from cluster 4 seem to possess a relative high expression in roots under control conditions, and those from cluster 7 seem to possess a relative high expression in leaves under control conditions. DIR genes from both clusters are down-regulated in response to osmotic stress. On the other hand, we also recovered a homolog of the ScDir gene (GenBank: JQ622282.1) from the sugarcane variety FN39 (DIR38 in cluster 5). The expression of ScDir from FN39 has been

decreasing the expression of genes involved in sucrose and starch metabolic processes, cell wall biogenesis, cellulose biosynthesis, anion transport, and light response. A handful of the genes found by Pereria-Santana and cols. Are presented along with their expression profiles in the heat map of **Figure 5A**. Because of the well-defined expression pattern of some of these genes, they could prove to be useful as expression markers in the response of 'MEX69290' to osmotic stress. For example, ABA 8-hydroxylase 3, Isoflavone 2-hydroxylase, LEA 14A, and NAC TF 25 showed clear patterns of up-regulation. In fact, in our laboratory further expression and functional analyses are currently being carried out regarding this NAC TF25 gene. Conversely, Bidirectional sugar transporter SWEET11, Cellulose synthase E6, and Sugar transporter ERD6 16 showed clear patterns of down-regulation. These down-regulated genes are also interesting, not just because of their responsiveness to osmotic stress but also due to their involvement in sucrose metabolism. The engineering of these genes might increase biomass production in sugarcane and tolerance to osmotic stress simultaneously. Furthermore, many TFs known to play important roles in the stress responses of plants, i.e. HSF, ZN, bZIP, WRKY, NAC, and MYB, were found in abundance in the total assembly of the 'MEX69290' transcriptome (**Figure 5B**). Even when some of these TF families seemed underrepresented (like NAC and MYC), they still provide a useful benchmark to conduct phylogenetic, expres-

**Figure 5.** Selected DEGs in response to osmotic stress and abundance of major TF families and Dirigent protein family in sugarcane 'MEX69290' transcriptome. (A) Expression profile of 20 selected DEGs in leaves and roots of sugarcane 'MEX69290' plantlets submitted to PEG-8000 treatment during 0, 24, 48, and 72 hours. Data was obtained from the work of Pereira-Santana and cols [52]. The heat map was generated with the ComplexHeatmappackage v1.14.0 [52] in R v3.4.1 [53]. (B) Abundance of major stress-related TF families and Dirigent protein family in arabidopsis, rice, sorghum, and sugarcane. The results were obtained by means of HMM searches using the profiles of the HSF (PF00447), ZF (PF00096), bZIP (PF00170), WRKY (PF03106), NAC (PF02365), MYB (PF00249), and Dirigent (PF03018) proteins obtained from the Pfam database (http://pfam.xfam.org) [54]. For this analysis the complete predicted proteomes (primary transcripts only) of arabidopsis, rice, and sorghum were obtained from Phytozome v. 12 [55]. Sugarcane predicted protein dataset was obtained from the transcriptome assembly of Pereira-Santana and cols [52] HMM searches were performed using HMMER3 v3.1b2 (http://hmmer.org/) and set to a cut-off e-value of 1e-05 and a score above the inclusion threshold of

sion, and functional analysis.

98 Plant, Abiotic Stress and Responses to Climate Change

each HMM profile.

**Figure 6.** Differential expression in response to osmotic stress of 48 Dirigent proteins found in sugarcane 'MEX69290' transcriptome. The 48 Dirigent sequences from sugarcane were grouped according to their expression profiles in 7 clusters (1–7). Data was obtained from Pereira-Santana and cols [52]. Heat map and sequence clustering were generated with ComplexHeatmap v1.14.0 [53] in R v3.4.1 [54] using the "euclidean" distance method and "complete" clustering method.

reported to be up-regulated in response to H2 O2 , NaCl, and PEG treatment [59]. Furthermore, its heterologous expression in *Escherichia coli* increases the bacterial host's tolerance to NaCl and PEG [59]. The homolog of this gene in 'Mex 69-290' was slightly up-regulated in leaves, but down-regulated in roots (**Figure 6**, cluster 5). All of these mentioned DIR genes from sugarcane 'MEX69290' are interesting because they show differential expression patterns in leaves and roots in response to osmotic stress. However, their functional roles in osmotic stress tolerance and biomass accumulation still need to be experimentally analyzed. In summary, in the absence of a complete sequenced genome for sugarcane, high-throughput sequencing technologies applied to the elucidation of elite cultivars' transcriptome profile are one of the most valuable resources for the identification of genes involved in both stress tolerance and biomass accumulation, which are important agronomic traits to face global climate change.

after *A. tumefaciens* transformation via axillary shoots [67]. These latter two protocols require

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In contrast, a genetic transformation protocol using *A. tumefaciens* has been developed in our laboratory (in the process of obtaining patent) where *in vitro* basal micro-shoots of MEX69290 cultivars underwent the insertion of the CpRap2.4b gene from the AP2/ERF transcription factor family, and out of cDNA of papaya stressed at 40°C. This genetic transformation protocol requires only 20 minutes and has a contamination rate of 0%, as well as a 21-day seedling regeneration rate. Our results showed a 70% survival in the first subculture and 100% in the second subculture with Kanamycin; similar results were reported by Manickavasagam regenerating transgenic seedlings using micro axillary outbreaks out of field plants [67], with a very laborious genetic transformation system and with 50% survival in the first crop. In addition, this work would be the second in sugarcane to report a gene of the AP2/ERF family of transcription factors inserted in sugar cane, the other work is the one reported by Reis et al. where they over expressed AtDREB2A CA (constitutive activity) in sugar cane [68]. In the transformed sugarcane seedlings generated by the genetic transformation protocol that was developed in our laboratory, the presence of the GFP was observed at the fluorescent emission of 395–475 nm, which indicates that the seedlings are

It should be clarified that the functionality of the CpRap2.4b gene belonging to the (AP2/ ERF) transcription factors family was tested in tobacco plants, which were segregated to obtain F2 plants and were then subjected to water stress (drought) conditions to evaluate

**Figure 7.** GFP fluorescence of different plant leaves of sugar cane var. MEX69290. (A) Segment of wild leaf in visible light. (B) Wild leaf segment with emission at 509 nm. (C, E and G) Transgenic plants 1, 2 and 3 in visible light. (D, F and

a time lapse between 3 and 6 months to generate seedlings.

transformed (**Figure 7**).

H) Transgenic plants 1, 2 and 3 with emission at 509 nm.

their function.
