Study On Physiological And Molecular Mechanism Of Cold Resistance Enhanced By Exogenous ABA Application In Sugarcane

Sugarcane is the most important sugar crop in China and the world, which originated in tropical and subtropical areas and is a thermophilic crop. Extreme weather occurred frequently in worldwide that caused serious cold and/or frost damage in recent years, resulting in enormous losses in sugarcane production. The main measures to decrease the losses caused by cold and frost damage in commercial sugarcane production are selecting cold resistant sugarcane varieties, improving cultivation management and chemical regulation etc. ABA plays an important role in plant growth especially in adverse enviroment. In the present study, seedcane setts were grown in soil culture and solution culture to investigate the physiological and molecular mechanism of cold resistance enhanced by ABA application in sugarcane with strong cold resistant variety GT28and weak cold resistant variety YL6using real-time PCR,2-DE and MS. The main results were as follows.1. Effects of exogenous ABA on physiological and biochemical of sugacane settlings under cold stressLow temperature caused significant changes in the physiological and biochemical characteristics in leaves of sugarcane plants. The results showed that, under the cold stress condition, the plasma membrane permeability and contents of MDA and proline increased; for photosynthesis related parameters, Pn, Gs, Tr decreased while Ci increased; for fluorescent parameters, Fv/Fm, qP and ΦPSII increased, but Fo and NPQ decreased; for endogenous hormones, GA and ZR content decreased while ABA content increased, and the ratios of ABA/GA, ABA/IAA, ABA/ZR increased. Genotypic differences in response to the contents of proline, ABA and GA, and the ratio of ABA/GA exist between strong cold resistant variety GT28and weak cold resistant variety YL6under cold stress. The contents of proline and ABA and the ratio of ABA/GA are higher and the content of GA is lower in the strong clod resistant variety, which is the vital physiological basis that caused two sugarcane varieties with different cold resistance. It is concluded that the ABA application effectively alleviated the adverse effect of low temperature on the plant growth, which kept the relative stability of cell membrane, Pn, PS Ⅱ and chlorophyll, decreased the contents of MDA and GA, and increased the contents of proline and ABA and the ratio of ABA/GA, thereby increased the cold resistance of sugarcane settlings.2. Effect of ABA and its biosynthesis inhibitor on the activities of antioxidant enzymes in sugarcane settling under cold stressThe activities of antioxidant enzymes were increased under cold stress. After ABA application, the content of ABA and the activities of SOD, APX, CAT, POD and GR increased in sugarcane settlings, while the contents of O2-, H2O2and MDA decreased. After tungstate (biosynthesis inhibitor of ABA) application, the result was contray. It is concluded that ABA and its biosynthesis inhibitor have an important regulatory role to the antioxidant protection system in sugarcane settling under cold stress. The cold resistance in sugarcane enhanced by ABA application under cold stress may be associated with the induction of antioxidant protection system.3. Effects of exogenous ABA on protein expression in sugacane settlings under cold stressThe protein expression was changed in settling leaves under cold stress. The results of2-D gel electrophoresis analysis showed that48differential protein spots were detected under cold stress and cold stress plus ABA application conditions in two sugarcane varieties, and29of them were successfully identified through mass spectrometry. The29proteins were involved into seven categories. Seven proteins participate in photosynthesis, accounted for24.1%, including photosystem Ⅱ stability/assembly factor, chlorophyll a-b binding protein, ferredoxin-NADP-reductase, ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit, photosystem I reaction center subunit Ⅱ,23kD polypeptide of photosystem II and chloroplast-localized Ptr ToxA-binding protein;7proteins participate in defense responses, accounted for24.1%, including glyoxylase, ascorbate peroxidase, thioredoxin peroxidase, thioredoxin M-type, quinone reductase, super-oxide dismutase and glutathione S-transferase;6proteins participate in protein processing, accounted for20.7%, including20S proteasome,3OS ribosomal protein,60S acidic ribosomal protein, FKBP type peptidyl-prolyl cis-trans isomerase, BRII-KD interacting protein and peptidyl-prolyl cis-trans isomerase;6proteins participate in metabolism, accounted for20.7%, including aspartate aminotransferase, chloroplastic aldolase, thiamine biosynthesis, triosephosphate isomerase, fructose1,6-bisphosphate aldolase precursor and ATP synthase delta chain;1protein participates in transcription, accounted for3.4%, which is eukaryotic translation initiation factor5A;1protein participates in cell growth an division, accounted for3.4%, which is an auxin-binding protein; and1protein is unclassified, accounted for3.4%, which is a hypothetical protein. Gene expression analysis of10differential proteins done by quantitative real time PCR showed that the mRNA level was not correlated well with the protein level. It was found that cold stress mainly affected photosynthesis and antioxidant protection system through changes in protein expressions in sugarcane, which is consistent with result of physiological analysis. After ABA treatment, stabilized the photo synthetic system and strengthened the role of antioxidant protection through a series of protein processing and folding, thereby increased the cold resistance of sugarcane settlings.4. Cloning and expression analysis of cold resisitance genesFive cold resistance genes were cloned by RT-PCR and RACE. A full length of SoAPX gene was obtained and the cDNA was1045bp. A full length of SoGST gene was obtained and the cDNA was912bp. A full length of SoASR gene was obtained and the cDNA was753bp. A full length of SoFNR gene was obtained and the cDNA was1335bp. A full length of SoDHN gene was obtained and the cDNA was578bp. All the genes were expressed in Prokaryon and transformed into E. coli and expressed successfully. The mRNA expressions of SoAPX, SoGST, SoASR, SoFNR and SoDHN were analyzed at the transcriptional level. The results showed that, low temperature caused significant differences in gene expression and regulation of cold resistance in two sugarcane plants. SoAPX gene is highly expressed in strong cold resistant variety GT28longer than weak cold resistant variety YL6, SoGST gene is started in stong cold resistant variety GT28faster than weak cold resistant variety YL6, SoASR gene is up-regulated in stong cold resistant variety GT28and the highest expression level in1day after cold stress, but it is down-regulated in weak resistant variety YL6, SoDHN gene significantly increased in stong cold resistant variety GT28and the highest increase reaches by1200.6%, while the highest increase in weak resistant variety YL6is only38.9%. It indicated that, the differential expression of sugarcane cold-related gene is directly impacted in the cold resistant genotypes. After ABA pretreatment, the expressions of the five genes were induced in different extent, which refleted that the role of ABA signal transdution was enhanced, the capacity of antioxidant defense was increased, the structure of membrane was relatively stable, thereby increasing the cold resistance of sugarcane settlings. The induction and regulation of cold resistance related genes by ABA application is an important molecular basis to improve the cold resistance in sugarcane.