Materials and Methods

Plant material

Setts of sugarcane (Saccharum sp.) plants of cultivar, Co86032 in the form of entire transverse nodal culm sections, bearing a single intact axillary bud was collected from Sugarcane Breeding Institute, Coimbatore, Tamil Nadu, India. The collected plant materials were maintained in plastic pots containing soil: vermiculite: organic fertilizer, 3:2:1, (w/w/w) under greenhouse conditions (16/8-h photoperiod; 26 °C). The sugarcane plantlets developed from the axillary bud of the setts were used for stress induction.

To identify sucrose synthase (SuSy) gene homologues in sugarcane by computational methods, the steps indicated in were followed and specific primers for the SuSy genes were designed using Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/primerinfo.html) [7].

Stress induction in sugarcane plantlets

The setts of sugarcane maintained in greenhouse conditions germinated after 8 days of planting. The plantlets were divided into three groups of three plants each; one group served as control, the second group was induced with drought stress and the third group was induced with salt stress. To develop water-deficit stress, watering to the test plants were withheld during the tillering stage (30 - 60 days after planting) for the designated period. For salt stress, test plants were grown in soil containing 2 % (w/w) of NaCl. The control plants were maintained without salt stress and received normal watering throughout the experiment. The uppermost fully expanded leaf samples were collected at 0, 4, 8, 12, 16 and 20 Days after Stress (DOS) from control, water-deficit and salt stressed plants. The collected leaf tissues were quick-frozen and stored at -80 °C until RNA extraction. RNA was extracted from the leaf samples using RNA extraction and purification kit (Sigma Aldrich) as per manufacturer’s instructions and cDNA was synthesized.

Amplification of the SuSy gene coding sequence

The cDNA synthesized from the leaf samples were subjected to PCR amplification. A total of 9 primers were designed for SuSy gene using Primer-BLAST were used for amplification of the synthesized DNA (Both Control and stress induced) using PCR (Millipore) as per manufacturer’s instructions. The unincorporated PCR primers and dNTPs were removed from PCR products by using Montage PCR Clean up kit (Millipore). The PCR product was sequenced using the primers. Sequencing reactions were performed using ABI PRISM^® BigDye^TM Terminator Cycle Sequencing Kits with AmpliTaq^® DNA polymerase (FS enzyme) as per manufacturer’s instructions (Applied Biosystems). The sequenced genes were subjected to Bioinformatics analysis.

Bioinformatics Protocol

1. The 16s rRNA sequence was blast using NCBI blast similarity search tool. The phylogeny analysis of query sequence with the closely related sequence of blast results was performed followed by multiple sequence alignment.

2. The program MUSCLE 3.7 was used for multiple alignments of sequences [9]. The resulting aligned sequences were cured using the program Gblocks 0.91b. This Gblocks eliminates poorly aligned positions and divergent regions (removes alignment noise) [10]. Finally, the program PhyML 3.0 aLRT was used for phylogeny analysis and HKY85 as Substitution model.

3. PhyML was shown to be at least as accurate as other existing phylogeny programs using simulated data, while being one order of magnitude faster. PhyML was shown to be at least as accurate as other existing phylogeny programs using simulated data, while being one order of magnitude faster. The program TreeDyn 198.3 was used for tree rendering [11].

4. The protein sequences of the identified genes were further subjected to ProtFun 2.2 server (http://www.cbs.dtu.dk/services/ProtFun/) to predict the function of the identified sequences. The sequences of the identified motifs were given as the input in the FASTA format for the predicting their functions. The parameters were set as default.

Expression analysis of the identified SuSy gene using RTPCR during abiotic stress condition

To validate the predicted function and expression pattern of the predicted SuSy genes, RT-PCR was performed by following the established procedures using the isolated RNA samples and designed primers [12]. Real Time - PCR (RT-PCR) amplification was performed using a MX3005P thermocycler (Stratagene/ Agilent Technologies) as per standard protocol. The RT reaction mix without reverse transcriptase served as a negative control. For internal control, primers NtActin_FP: 5’-ATGGCAGACGGTGAGGATATTCA-3’ and NtActin_RP: 5’-GCCTTTGCAATCCACAT CTGTTG-3’ was used for amplification.

The dissociation curves were generated at 95 °C to verify the PCR specificity after completion of PCR cycling. Quantities of RNA accumulation levels were calculated as RQ values using the comparative cycle threshold (CT) (2-ΔΔCt) method. Dissociation curve/ Melt curve is generated by increasing the temperature from 55 °C to 95 °C in tiny increments and monitoring the intensity of fluorescence at each step. All qPCR reactions were performed in three technical replicates.

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Results and Discussion

Comparative genomics studies have been used to analyze SuSy gene families in various plant species, including citrus, tobacco, poplar, and cotton [13,14]. Since the complete genome of sugarcane is not available as it is polyploidy in nature and is very difficult to determine the complete CDS regions, the only option left behind is the usage of EST sequences. According to the computational analysis, SuSy gene homologues were searched in sugarcane nucleotide sequences. SuSy genes (896 genes) collected from plant transcription databases were subjected to local BLAST against the 3268 sugarcane transcription factor genes involved in sucrose synthesis. A total of 75 putative sequences were identified after the local BLAST analysis. After redundancy removal, the individual sequences were subjected to contig assembly using the CAP3 program which resulted in 26 candidate sequences. The resulting sequences were subjected to PROSITE, MEME and ProtFun analysis program to confirm their characteristic domain and its function [15].

Abiotic stress causes many changes in the morphological, physical and biochemical properties of a plant which may lead to decrease in the regular metabolic activities of the plant (Figure 2). The morphological changes start with decrease in the chlorophyll content, curling of the tips of the leaf, dryness, yellowness of the leaf and finally to turning the leaf to brown color loosing the rigidity. These changes occur slowly at a constant rate under salt stress as well as drought stress conditions [16]. The additional changes that can be observed in drought stress is change in the osmotic conditions of the plant leading to decrease in the relative water content of the plant. This change affects the photosynthesis of the plant which is connected with the sucrose synthesis and transport [3], since sucrose in synthesized during the photosystem II [17], gets transported to the culm of the sugarcane and accumulates there. The quantity of accumulated sucrose decides the length and thickness of the stalk of the sugarcane which decides its commercial value.

Figure 2: Morphological changes observed in plants during the course of drought and salt stress induction. (a) Control plant on 0^th day. (b) On 4^th day. (c) On 8^th day. Yellowness on the leaf was observed. (d) On 12^th day. Dryness and curling of the leaves were observed. (e) On the 16^th day. Almost 50% of the leaves were dry. (f) On the 20^th day. The entire plant was dry and did not survive after 20 days.

Of the 9 primers designed, only one gave a good result which is shown in Table 1. So, this primer was taken for further expression analysis in the stress induced samples. The amplified PCR product was quantified, purified and sequenced to obtain the complete sequence of the amplified gene (Figure 3). From the Figure 3, it is clear that the primer designed for analyzing the synthesis of sucrose synthase during abiotic stress conditions gave a good amplification the possibility of expression of the SuSy gene during stress condition also. The nucleotide sequence thus obtained was subjected to Bioinformatics analysis. Figure 4-8 depicts the results of Bioinformatics analysis which confirms the isolated gene is SuSy gene belonging to MYB transcription factor family.

Table 1: Primer Details.

Figure 3: PCR amplification of DNA from stress induced samples using designed primer.

C - Control; D4 - Day 4, D8 - Day 8, D12 - Day 12, D16 - Day 16, D20 - Day 20 of stress induction. Drought - DNA isolated from drought stress induced leaf samples; Salt - DNA isolated from salt stress induced leaf samples.

Figure 4: Amplified SuSy gene sequence.

Figure 5: Translation of SuSy gene in Frame 1.

Figure 6: Phylogenetic analysis of the identified SuSy sequence.

Figure 7: MEME motif determination and Prosite results.

Figure 8: ProtFun 2.2 Server results of the predicted SuSy gene.

Expression patterns of stress inducible genes are very complex. Some genes respond to drought, salt and cold very rapidly, whereas others are induced slowly after the accumulation of hormonal signals. RT-PCR was carried out using primers specific to SuSy gene for validating its predicted biological function. In this study, Sc25S rRNA was used as reference gene for RT-qPCR experiment [18]. The SuSy transcripts expression level at different stages of stress is indicated in Figure 9. From the above figure, it can be understood that the SuSy gene got expressed at almost same level during the initial period of stress (0-8 DOS) and from 12^th DOS, the level of expression started decreasing. Interestingly, on the 20^th DOS, it is found that the SuSy gene got under expressed in the drought condition. This can be correlated with the non availability of water on the 20^th DOS which seriously affects the photosynthesis of the plant. Since the light reaction of photosynthesis cannot occur in the absence of water, the synthesis process of sucrose drastically decreased as the gene was not able to express itself.

Figure 9: Expression of the SuSy gene at different stages of abiotic stress.

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Summary

The results of the study can be summarized as follows: 3268 sucrose synthase genes from sugarcane collected from transcription factor databases were analyzed computationally and 26 unique candidate genes were shortlisted. These 26 genes were overlapped for complete CDS formation and 9 gene specific primers were designed. Saccharum officinarum Co86032 was maintained in the green house condition and abiotic stress (Salt stress and Drought) was induced; leaf samples were collected at 4 days interval, RNA was isolated, cDNA synthesized and amplified using gene specific primers. Only one primer gave good amplification in all the samples and so that amplified gene was taken for sequencing. The sequenced gene and identified protein sequence was subjected to various Bioinformatics analysis to identify the motifs and its functional role. The gene was taken for RT-PCR analysis and the level of expression of the gene wa analyzed in the stressed as well as control plants. The identified SuSy gene was expressed at a reasonable level during the initial period of stress but the expression level started decreasing after 12^th DOS. It can be inferred that this gene which got expressed during the initial period has the capacity to withstand the abiotic stress to certain level. If this candidate gene is taken for overexpression studies, it can serve as a potential candidate for developing transgenic sugarcane which contains the overexpressed SuSy gene incorporated into its genome which will render resistance for the plant against abiotic stress. Also, during the stress condition, if the expression level of a gene remains standard, the gene will be able to induce the synthesis of sucrose. Thus, this study will form a basis for gene identification and expression and developing stress resistant sugarcane plant which accumulates high sucrose content even under stress conditions and development of transgenic plants containing overexpressed gene will be beneficial for industrial production of sugar.

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Acknowledgements

The authors are thankful to UGC-SERO for funding this work.

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