Sample collection

The plant materials for this research were collected from Sugarcane Crop Research Institute Mardan, Khyber Pakhtunkhwa Pakistan. Three commercially grown sugarcane cultivar namely SPSG 394, CP77_400, CP85-1491 based on resistance to cold stress and wild type S. spontaneum L. was selected as control from cold region Murree, Khyber Pakhtunkhwa Pakistan. The sugarcane buds were collected during maturing stage and harvested in the research field of Genetics Department Hazara University, Mansehra (Table 1).

DNA Extraction

The genomic DNA was extracted from the fresh leaves of sugarcane cultivars and wild relative S. spontaneum L. by using modified CTAB methods  [16].

The fresh leaves of sugarcane cultivars and S. spontaneum L. were crushed in liquid nitrogen with the help of pestle and mortar, and crushed samples (0.25 mg) was taken in 2 ml Eppendorf tube, and the pre-heated CTAB buffer (900µl) was added to each tube, and then incubated at 56°C for 24 hours. After incubation, 500µl mixture (composition) of Phenol Chloroform (PCI) was added to each tube and the samples were centrifuged at 8000xg for 20 minutes. After centrifugation, the clear supernatants were transferred into fresh Eppendorf tubes and 500ul of cold isopropanol were added to the supernatants. The samples were kept in -20°C for 20 minutes and then centrifuged at 8000xg for 20 minutes. After centrifugation, the supernatant was discarded, and the pellet was washed by adding 500µl of 70% ethanol. After washing, the samples were left at room temperature and 60µl of TE buffer were added to each tube and vertex well. The DNA quality and quantity was checked on 1 % Agarose gel.

Gel Electrophoresis

The extracted genomic DNA was checked on 1% agarose gel. One gram agarose powder was dissolved in (98 ml dH2O and 2 ml 50x TAE (Tris-acetate-EDTA), the mixture was boiled in a conical flask. Till the agarose dissolved completely, 25µl ethidium bromide was added and then gel was cast in a gel tray with a comb. After solidifying, gel was placed in the gel tank containing 50x TAE. Five µl of DNA from each sample was taken, mixed with 2µl loading dye and loaded in the wells. The gel was then run at constant voltage of 75 volts for approximately one hour and 30 minutes. The gel was observed under UV light using “Uvitech” gel documentation system.

Selection of Genes and Primer designing

Several cold responsive genes were selected from public database and reported literature of Andropogoneae tribe. Further, primers were designed according to the conserved regions of the genes (Table 2) and these primers were synthesized from Macrogen Korea.

PCR amplification

For the amplification of twenty three cold responsive (COR) genes in the genome of sugarcane cultivars and S. spontaneum L. Thermo scientific PCR kit (Catalog #EP0402) was used and followed the instruction of the manufacturer. The PCR amplified products were further confirmed by 1.5% gel and the amplicon size was compared with 1Kb DNA marker. The 23 cold tolerance genes (COR) were further confirmed in genomic DNA of sugarcane cultivars and Saccharum spontaneum L. by gel electrophoresis. Although for further confirmation the samples were sent to the Beijing Genomic, Institute China for sequencing. The optimized sequences of sugarcane cultivars and S. spontaneum L. were analysed by using NCBI protein and nucleotide BLAST. The sequences of cold tolerance genes (COR) of sugarcane cultivars and S. spontaneum L. were also analysed to study the conserved domains in the conserved domain (CDS) search NCBI.

Results

DNA Confirmation

Whole genomic DNA was extracted from fresh leaves of three commercially grown sugarcane cultivars and a wild relative S. spontaneum L. from Pakistan either resistance or sensitive to cold stress. DNA was analysed by electrophoresis prior to use as a template in PCR reactions. The high qualities DNA free from impurities i.e. RNAs, protein which could hinder PCR amplification, was purified (Fig. 1).

Confirmation of targeted genes

For PCR optimization of target DNA using cold tolerance genes (COR) various condition were used. The conditions were also modified by changing the annealing temperature and annealing time. The amplified PCR product of cold tolerance gene (COR) was confirmed by 1.5% Agarose gel. The cold responsive genes were further analysed in the genome of selected sugarcane cultivars and wild relative S. spontaneum L. through PCR and recorded on Agarose gel electrophoresis (AGE). The analysis of selected cold responsive genes (COR) showed 100% amplification, among 23 genes 16 were successfully amplified in all selected sugarcane cultivars and S. spontaneum L. while 7 genes were found unique (Table 3).

For further investigation, the amplified COR genes were selected for sequencing based on presence of COR genes in the genome of investigated sugarcane cultivars and wild relative S. spontaneum L. Furthermore, the successful sequences of COR genes in selected sugarcane cultivars and S. spontaneum L. were further analysed by using NCBI Protein BLAST (Basic Local Alignment Sequence tool) for comparison of already available data. Further, analysis of the successful sequences of COR genes showed that these COR genes encode cold and other abiotic stress responsive protein (Table 4).

Sequence Analysis

The sequences of the selected cold tolerance genes (COR) were also analysed using NCBI Conserved Domain search (CDS). Three significant conserved domains were detected, during conserved domain analysis of CBF2, WCOR14 and WCS66 genes retrieved from SPSG-394, CP-851491 and S. spontaneum L. Conserved domain (Gypsy type transposon) was detected during the analysis of CBF2 gene sequence. These families of plants genes revolved close association with gypsy type transposon and have an important role in the regulation of genes. The conserved domain (Zinc binding region) was detected in COR47 gene sequences, and this domain also called zinc finger acts as small protein motif and characterized as binding region for various molecules and also helps in order to stabilize the protein folding. Furthermore, conserved domain (aspartate proteases) detected by WCS66 gene sequence proteases (peptidases or proteinases) are a large category of enzymes that catalyze the hydrolysis of peptide bond. The current research work was conducted for the first time in the Khyber Pakhtunkhwa region of Pakistan for the identification of cold tolerant commercially grown sugarcane cultivars. The PCR based analysis of cold tolerance genes in selected sugarcane cultivars and wild relative S. spontaneum L. showed positive and significant conclusion and results. Sugarcane plant during cold stress reprogram their gene expression through transcriptional, translational and posttranslational mechanism, CBF transcriptional factors and other cold responsive genes plays crucial role in cold stress.  Based on results of cold tolerance genes in selected sugarcane cultivars and wild relative S. spontaneum L. these cold responsive genes showed 100% amplification, while among the investigated sugarcane cultivars SPSG-394 and CP-851491 showed significant results most of the cold responsive genes were amplified in their genome.

Figures & Tables

The response of plants to cold stress is a complex process involved many physiological and biochemical modifications. The expression of genes and protein metabolites in the response to cold stress has been reported [16].  In this study transcription factors, namely CBF1, CBF2 and CBF3 were investigated in selected sugarcane cultivars and wild relative S. spontaneum L. through PCR and further confirmed by sequencing. Moreover, CBF1 gene was observed in cold tolerant cultivar CP-851491 and wild relative S. spontaneum L. further, CBF2 gene was optimized in sugarcane cultivars CP-851491, SPSG-394 and wild relative S. spontaneum L. while the CBF3 gene was optimized in all sugarcane cultivars and wild relative S. spontaneum L (Table 3). In this study, WCOR14 gene was optimized in all selected sugarcane cultivars and wild relative S. spontaneum L. while the sequence result of WCOR14 showed that this gene encodes COR14 protein (Table 4).

Further, cold responsive gens example includes COR15a and COR15b, are expressed during cold [8]. In this study COR15a and COR15b were amplified in all sugarcane cultivars and S. spontaneum L. Moreover, wheat cold tolerance genes WCOR 726, WCOR80, WCOR410, WCS 120, and WCS66 that contributes to freezing tolerance [11]. In this study, WCS66, WCS120, WCOR726, WCOR 615 and WCOR80 were amplified in all sugarcane cultivars and wild relative S. spontaneum L. while the WCS66 gene sequence analysis showed conserved domain (aspartate proteases) during NCBI CDS search. The cold responsive genes C17, 85KDA, COR410, COR 6.6, COR 47 and COR 78/RD29 are expressed during cold, dehydration or ABA stress. The expression of these genes has shown to be critical for both cold acclimation and chilling tolerance in plants [10]. In this study the C17 and 85KDA genes were amplified in all sugarcane cultivars and wild relative S. spontaneum L. and encode proteins like cold shock protein CS66-like and Phosphoprotein ECPP44-like, these protein help in cold temperature. While COR47 encodes protein, hypothetical protein, which help in Calvin cycle catalyzing the carbon fixation and also help in plant metabolism, and COR78 encode small GTPs protein which helps in regulating the function of other protein, while COR6.6 encode KIN2 which helps in cold temperature and other abiotic stresses.

The current study was conducted to investigate the cold responsive genes in Pakistani sugarcane commercially grown cultivars. Our results revolved that many genes are involved in sugarcane during cold stress. Although in this study the genes were confirmed by PCR and DNA sequencing, moreover conserved motifs were also studied. We recommend more study on large scale using transcriptomic approaches could be more significant for the sugarcane industry of Pakistan.

Author Contributions

All authors contributed equally to this study and manuscript.

Conflict of Interest

The authors declare that there is no conflict of interest.

References

1. Rehman US, Muhammad K, Que Y, Rehman AU, Novaes E, Khan S. Presence of seventeen genes potentially involved in cold tolerance in sugarcane and Saccharum spontaneum genotypes. International Journal of Biosciences, (2019); 14(1): 346- 355.
2. Vilela MDM, Del Bem LE, Van Sluys MA, de Setta N, Kitajima JP, Cruz GMQ, Sforça DA, de Souza AP, Ferreira PCG, Grativol C and Cardoso-Silva CB. Analysis of three sugarcane homo/homeologous regions suggests independent polyploidization events of Saccharum officinarum and Saccharum spontaneum. Genome Biology and Evolution, (2017); 9(2): 266-278,
3. Moore PH 1987. Breeding for stress resistance. In: Heinz DJ, Sugarcane Improvement through Breeding ed., Elsevier, Amsterdam. pp 503-542.
4. Du YC, Nose A, Wasano K. Thermal characteristics of C4 photosynthetic enzymes from leaves of three sugarcane species differing in cold sensitivity. Plant Cell Physiology, (1999); 40(3): 298-304, 19998.
5. Que Y, Su Y, Guo J,  Wu Q, Xu L. A global view of transcriptome dynamics during Sporisorium scitamineum challenge in sugarcane by RNA-Seq. PLoS One, (2017); 9(8): e106476.
6. Tai PYP and Lentini RS 1998. Freeze damage of Florida sugarcane, University of Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, EDIS.
7. Gilmour SJ, Sebolt AM, Salazar MP, Everard JD and Thomashow MF. Overexpression of the Arabidopsis CBF3transcriptional activator mimics multiple biochemical changes associated with cold acclimation. Plant physiology, (2000); 124(4): 1854-1865.
8. Artus NN, Uemura M,  Steponkus PL, Gilmour SJ, Lin C and Thomashow MF. Constitutive expression of the cold-regulated Arabidopsis thaliana COR15a gene affects both chloroplast and protoplast freezing tolerance.  Proceedings of National Academy of Science, (1996); 93(23): 13404-13409.
9. Monroy AF, Castonguay Y, Laberge S, Sarhan F, Vezina LP and Dhindsa RS. A new cold-induced alfalfa gene is associated with enhanced hardening at subzero temperature. Plant Physiology, (1993); 102(3): 873-879.
10. Thomashow MF. Plant cold acclimation: freezing tolerance genes and regulatory mechanism. Annual Review of Plant Biology, (1999); 50: 571–59916.
11. Houde M, Dhindsa RS, Sarhan F 1992. A molecular marker to select for freezing tolerance in Gramineae.  Mol. Gen. Genet. 234(1): 43-48.
12. Rehman SU, Que Y, Khan S,  Novaes E, Inamullah and Muhammad K. Molecular Study Of CBF And WCOR14 Genes In Selected Sugarcane Cultivars and Its Wild Relative Saccharum spontaneum L. Journal of Animal and Plant Sciences, (2020); 30(4): 913-922
13. Yamaguchi-Shinozaki K, Shinozaki K. A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low temperature or high-salt stress. Plant Cell, (1994); 6: 251.264.
14. Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S, Yamaguchi-Shinozaki K, Shinozaki K. Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought-and low-temperature-responsive gene expression, respectively, in Arabidopsis. The Plant Cell, (1998); 10(8): 1391-1406.
15. Medina J, Bargues M, Terol J, Pérez-Alonso M, Salinas J. The Arabidopsis CBF gene family is composed of three genes encoding AP2 domain-containing proteins whose expression is regulated by low temperature but not by abscisic acid or dehydration.  Plant physiology, (1998); 119(2): 463-470.
16. Miura K, Furumoto T. Cold signaling and cold response in plants. International Journal of Molecular Sciences, (2013); 14(3): 5312-5337.