Nisachon JANGPROMMA, Akira SAITO, Tomohiro ARAKI, Prasit JAISIL, Patcharin SONGSRI, Sakda DADUANG

Molecular cloning and characterization in eukaryotic expression systems of a sugarcane cysteine protease inhibitor gene involved in drought tolerance

Cystatin responses in sugarcane plants under drought stress have not previously been reported. To test the hypothesis that sugar cane cystatin can function as an osmotic stress tolerance gene, transgenic Pichia pastoris (GS115) and Saccharomyces cerevisiae (A2279) strains with the ability to express sugarcane cystatin were constructed. The osmotic stress tolerance of the transgenic yeasts was then evaluated, and it was found that the transgenic Pichia pastoris (GS115) and Saccharomyces cerevisiae (A2279) had increased growth and increased osmotic stress tolerance. To gain a greater understanding of the responses of sugarcane cystatin to drought, 1 drought-susceptible and 3 drought-tolerant sugarcane cultivars were grown in a greenhouse for 3 weeks, exposed to drought stress for 5 days, and rehydrated for 5 days. Semiquantitative RT-PCR was subsequently performed, and the results showed increased sugarcane cystatin gene transcription in stressed plants when compared to the control. There was a greater increase in the drought-tolerant versus the drought-susceptible cultivars. However, the mRNA levels decreased once the plants recovered from the drought conditions. Hence, this study shows that the expression of cystatin in sugarcane is involved in drought stress. This gene may also serve as a target for future breeding programs focused on stress tolerance in sugarcane.

Anahtar Kelimeler:

Key words: Cystatin, Saccharum officinarum, water deficit, semiquantitative RT-PCR

Molecular cloning and characterization in eukaryotic expression systems of a sugarcane cysteine protease inhibitor gene involved in drought tolerance

Keywords:

Key words: Cystatin, Saccharum officinarum, water deficit, semiquantitative RT-PCR,

PDF

___

Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389–3402.
Aydın S, Büyük İ, Aras ES (2014). Expression of SOD gene and evaluating its role in stress tolerance in NaCl and PEG stressed Lycopersicum esculentum. Turk J Bot 38: 89–98.
Barrs HD, Weatherley PE (1962). A re-examination of the relative turgidity techniques for estimating water deficits in leaves. Aust J Biol Sci 15: 413–428.
Bera SK, Chandrashekar AB, Patel S, SojitraVK, Maurya A (2014). Identification of stable sources for surrogate traits in Arachis glabrata and marker-trait association for tolerance to water deficit stress. Turk J Bot 38: 309–324.
Caruso G, Cavaliere C, Foglia P, Gubbiotti R, Samperi R, Lagana A (2009). Analysis of drought responsive proteins in wheat (Triticum durum) by 2D-PAGE and MALDI-TOF mass spectrometry. Plant Sci 177: 570–576.
Cellier F, Conéjéro G, Breitler JC, Casse F (1998). Molecular and physiological responses to water deficit in drought-tolerant and drought-sensitive lines of sunflower. Plant Physiol 116: 319–328.
Chetthamrongchai P, Auansakul A, Supawan D (2001). Assessing the transportation problems of the sugar cane industry in Thailand. In: Transport and Communications Bulletin for Asia and the Pacific. New York, NY, USA: United Nations, pp. 31–41.
Cregg JM, Cereghino JL, Shi J, Higgins DR (2000). Recombinant protein expression in Pichia pastoris. Mol Biotechnol 16: 23–52.
Cui M, Zhang W, Zhang Q, Xu Z, Zhu Z, Duan F, Wu R (2011). Induced over-expression of the transcription factor OsDREB2A improves drought tolerance in rice. Plant Physiol Biochem 49: 1384–1391.
Demirevska K, Simova-Stoilova L, Fedina I, Georgieva K, Kunert K (2010). Response of Oryzacystatin I transformed tobacco plants to drought, heat and light stress. J Agron Crop Sci 196: 90–
Diop NN, Kidric M, Repellin A, Gareil MD, Arcy-Lameta A, Thi ATP, Zuily-Fodil Y (2004). A multicystatin is induced by droughtstress in cowpea (Vigna unguiculata (L.) Walp.) leaves. FEBS Lett 577: 545–550.
Gesimba RM, Njoka E, Kinyua M (2004). Root characteristics of drought tolerant bread wheat (Triticum aestivum) genotypes at seedling stage. Asian J Plant Sci 3: 512–515.
González L, González-Vilar M (2001). Determination of relative water content. In: Reigosa Roger MJ, editor. Handbook of Plant Ecophysiology Techniques. Dordrecht, the Netherlands: Springer, pp. 207–212.
Grudkowska M, Zagdańska B (2004). Multifunctional role of plant cysteine proteinases. Acta Biochim Pol 51: 609–624.
Habib H, Fazili KM (2007). Plant protease inhibitors: a defense strategy in plants. Biotechnol Mol Biol Rev 2: 68–85.
Hassanzadeh M, Ebadi A, Panahyan-e-Kivi M, Eshghi AG, Jamaatie-Somarin S, Saeidi M, Zabihi-e-Mahmoodabad R (2009). Evaluation of drought stress on relative water content and chlorophyll content of sesame (Sesamum indicum L.) genotypes at early flowering stage. Res J Environ Sci 3:345–350.
Hoshmand AR (2006). Design of Experiments for Agriculture and the Natural Sciences. 2nd ed. New York, NY, USA: Chapman and Hall.
Huang Y, Xiao B, Xiong L (2007). Characterization of a stress responsive proteinase inhibitor gene with positive effect in improving drought resistance in rice. Planta 226: 73–85.
Jangpromma N, Kitthaisong S, Lomthaisong K, Daduang S, Jaisil P, Thammasirirak S (2010a). A proteomics analysis of drought stress-responsive proteins as biomarker for drought-tolerant sugarcane cultivars. Am J Biochem Biotechnol 6: 89–102.
Jangpromma N, Songsri P, Thammasirirak S, Jaisil P (2010b). Rapid assessment of chlorophyll content in sugarcane using a SPAD chlorophyll meter across different water stress conditions. Asian J Plant Sci 9: 368–374.
Jangpromma N, Thammasirirak S, Jaisil P, Songsri P (2012). Effects of drought and recovery from drought stress on above ground and root growth, and water use efficiency in sugarcane (Saccharum officinarum L.). Aust J Crop Sci 6: 1298–1304.
Karaba A, Dixit S, Greco R, Aharoni A, Trijatmiko KR, MarschMartinez N, Krishnan A, Nataraja KN, Udayakumar M, Pereira A (2007). Improvement of water use efficiency in rice by expression of HARDY, an Arabidopsis drought and salt tolerance gene. Proc Natl Acad Sci U S A 104: 15270–15275.
Liu X, Wang Z, Wang L, Wu R, Phillips J, Deng X (2009). LEA 4 group genes from the resurrection plant Boea hygrometrica confer dehydration tolerance in transgenic tobacco. Plant Sci 176: 90–98.
Makbul S, Saruhan Güler N, Durmuş N, Güven S (2011). Changes in anatomical and physiological parameters of soybean under drought stress. Turk J Bot 35: 369–377.
Masoud SA, Johnson LB, White FF, Reeck GR (1993). Expression of a cysteine proteinase inhibitor (oryzacystatin-I) in transgenic tobacco plants. Plant Mol Biol 21:655–663.
Massonneau A, Condamine P, Wisniewski JP, Zivy M, Rogowsky PM (2005). Maize cystatins respond to developmental cues, cold stress and drought. Biochim Biophys Acta 1729:186–199.
O’Farrell PH (1975). High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250: 4007–4021.
Oliva MLV, Carmona AK, Andrade SS, Cotrin SS, Soares-Costa A, Henrique-Silva F (2004). Inhibitory selectivity of cane cystatin: a recombinant cysteine peptidase inhibitor from sugarcane. Biochem Biophys Res Commun 320: 1082–1086.
Rampino P, Pataleo S, Gerardi C, Mita G, Perrotta C (2006). Drought stress response in wheat: physiological and molecular analysis of resistant and sensitive genotypes. Plant Cell Environ 29: 2143–2152.
Reis EM, Margis R (2001). Sugarcane phytocystatins: identification, classification and expression pattern analysis. Genetics Genet Mol Biol 24: 291–296.
Robertson MJ, Inman-Bamber NG, Muchow RC, Wood AW (1999). Physiology and productivity of sugarcane with early and midseason water deficit. Field Crop Res 64: 211–227.
Sambrook J, Russell DW (2001). Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor, NY, USA: Cold Spring Harbor Laboratory Press.
Silva MA, Jifon JL, Silva JAG, Sharma V (2007). Use of physiological parameters as fast tools to screen for drought tolerance in sugarcane. Braz J Plant Physiol 19: 193–201.
Soares-Costa A, Beltramini LM, Thiemann OH, Henrique-Silva F (2002). A sugarcane cystatin: recombinant expression, purification, and antifungal activity. Biochem Biophys Res Commun 296: 1194–1199.
Solomon M, Belenghi B, Delledonne M, Menachem E, Levine A (1999). The involvement of cysteine proteases and protease inhibitor genes in the regulation of programmed cell death in plants. Plant Cell 11: 431–443.
Songsri P, Jogloy S, Holbrook CC, Kesmala T, Vorasoot N, Akkasaeng C, Patanothai A (2009). Association of root, specific leaf area and SPAD chlorophyll meter reading to water use efficiency of peanut under different available soil water. Agric Water Manage 96: 790–798.
Terzi R, Sağlam A, Kutlu N, Nar H, Kadıoğlu A (2010). Impact of soil drought stress on photochemical efficiency of photosystem II and antioxidant enzyme activities of Phaseolus vulgaris cultivars. Turk J Bot 34: 1–10.
Turkan I, Bor M, Ozdemir F, Koca H (2005). Differential responses of lipid peroxidation and antioxidants in the leaves of droughttolerant P. acutifolius Gray and drought-sensitive P. vulgaris L. subjected to polyethylene glycol mediated water stress. Plant Sci 168: 223–231.
Van der Vyver C, Schneidereit J, Driscoll S, Turner J, Kunert K, Foyer CH (2003). Oryzacystatin I expression in transformed tobacco produces a conditional growth phenotype and enhances chilling tolerance. Plant Biotechnol J 1:101–102.
Xiong L, Wang RG, Mao G, Koczan JM (2006). Identification of drought tolerance determinants by genetic analysis of root response to drought stress and abscisic acid. Plant Physiol 142: 1065–1074.
Zhang X, Liu S, Takano T (2008). Two cysteine proteinase inhibitors from Arabidopsis thaliana, AtCYSa and AtCYSb, increasing the salt, drought, oxidation and cold tolerance. Plant Mol Biol 68: 131–143.