Functional Characterization Of GlyoxalaseI And Xyloglucan Endotransglycosylase In Rice (Oryza Sativa L.)

Methylglyoxal(MG)is a kind of cytotoxic metabolites produced in glycolysis,amino acid catabolism,normal metabolism of acetone and environmental adversity stress,and exists in microbes,yeast,animals and higher plants.Combined Methylglyoxal and ?-carbonyl aldehyde from glyoxal with protein and nucleic acid form covalent compound by saccharification in vivio.Then,the covalent compound generating fructosamine and highly glycosylated end is toxic to cells.Glyoxalase(GLY)pathway includes two intracellular enzymes,GLY1 and GLY2,which generally exist in various kinds of organelles and act throughout the whole life progress.The metabolism of methylglyoxal takes place mainly through S-D-Lactic acid glutathione formed by condensation between methylglyoxal and glutathione(GSH)under the action of GLYI.Then,S-D-Lactic acid glutathione could be hydrolyzed into non-toxic GSH and D-lactic acid under the action of GLYII.Thus the MG concentration and its physiological activity could be reduced.About 61 GLY I sequences were identified and reported among various organisms.GLY I is closely involved in cell mitotic process,and the enzyme activity has been seen as "biomarkers" of cell growth and division.Also,GLY I generally takes part in the progress of regulation of cell division and proliferation,microtubule assembly and activation of vesicles.In addition,GLY I was validated to be involved in responses to abiotic stress in the plant.And over-expression of GLY I gene in the plant enhances its resistance to salt and heavy metal.Relatively less number of glyoxal enzyme genes cloned from plants,and their functions other than salt resistance were poorly understood.In the present study,a rice gene OsGLYI10 was characterized through applying reverse genetics tools.Bioinformatics analysis showed that OsGLYI10 encodes a protein with 209 amino acids containing GLYI conservative domain residing in 49-171 amino acids,suggesting OsGLYI10 is rice GLY1 gene.The homology comparison showed that OsGLYI10 gene is highly homologous to GLY I genes from other species.OsGly I was detected by quantitative real-time PCR(qRT-PCR)in different organs of rice plant,including root,stem,leaf,leaf sheath,and spikelet.The higher expression level was shown in leaf and spikelet.Expression of OsGly I was induced in rice seedlings treated with 250 mM NaCl,25 mM ZnCl2,and 500 mM mannitol.Among these treatments,ZnCl2 induced the highest level of OsGly I,3.6-fold greater than non-treated seedlings,followed by NaCl,and then mannitol,which showed an increase with approximately 2.6 or 2.2-fold,respectively.To elucidate the stresses tolerance function of Os GlyI,the construct pHB-GlyI was introduced into wild-type rice cv.Nipponbare via Agrobacterium-mediated transformation to generate OsGlyI-overexpression transgenic plants.Three independent transgenic T2 lines were used to detect the expression level of OsGly I by q RT-PCR.The result showed that OsGly I was increased to 1.8,3.5 and 8.3-fold,respectively,in the three T2 transgenic OsGly I-overexpression lines than that in WT plants.The transgenic lines were also used to detect the activity of glyoxalase I.Consistently,the three T2 transgenic OsGlyI-overexpression lines indicated increased enzyme activity,1.1,1.3 and 1.4-fold,respectively,compared with WT plants.Stress tolerance analysis indicated that seedlings of transgenic lines displayed higher growth rates than WT in response to the various abiotic stresses(including salt,heavy metal and drought)and the MDA content was remarkably lower in transgenic lines compared to WT plants.Agronomic trait analysis indicated that no significant differences in the number of effective panicles per plant,panicle length,grain numbers per panicle,or 1000-grain weight were shown between WT and transgenic lines.However,the seed setting rate was remarkable higher in all the three transgenic T2 lines as compared to the WT.The higher seed setting rate might contribute to higher yields resulting from the transgene expression.Xyloglucan endotransglycosylase/ hydrolytic enzymes family is a large gene family.It encodes an enzyme involved in regulating cell wall elasticity and ductility,plant growth and development,stress responses,and other functions.The primary cell walls from commelinoid monocotyledons,which include cereals such as rice(Oryza sativa),have a number of major structural and compositional differences from those of dicotyledonous species and are termed type II cell walls.Most studies report that type II cell walls have relatively little xyloglucan,and the predominant glycan that cross-links cellulose microfibrils is instead of glucuronoarabinoxylan.The genome sequence of rice subsp.Japonica cv Nipponbare has been published by the International Rice Genome Sequencing Project.Analysis of the draft sequences of the rice genome has revealed a large rice XTH(OsXTH)gene family with 29 members,a number that,contrary to expectations,is similar to that of the At XTH gene family.This surprising finding raises interesting questions about the biological importance of XTHs in species with type II walls.A detailed understanding of regulation factions of OsXTH family members in rice cell wall structure,however,is lacking.We choose OsXTH19 for the present study.Expression of OsXTH19 gene was detected in different rice tissues,with highest express in spikelet and lowest express in leaf and leaf sheath.Its expression was enhanced by indole-3-acetic acid and gibberellic acid.Transgenic lines over-expressing OsXTH19 were obtained using Agrobacteriummediated gene transformation.Compared to wild-type plants,transgenic rice lines grew more rapidly under normal growth conditions,and was more sensitive to GA3 and IAA,suggesting that OsXTH19 is related to plant growth.In addition,the transgenic lines exhibited higher tolerance than wild-type under stress of 100 mmol·L-1 NaCl and contained decreased malondialdehyde in cell lipid peroxide,indicating overexpression of OsXTH19 gene could improve slat tolerance in rice by participating in the anti lipid peroxidation.Cellulose,hemicelluloses,silica content as well as the pull strength were significantly lower in the transgenic lines than in WT plants.The results demonstrated that transgenic lines overexperssing OsXTH19 resulted in reducting main ingredients of cell walls and relaxating the cell walls and consequently leading to pull strength decreased.Paraffin section analysis that,although the cell wall components have beend changed,no obvious changes on morphology were observed.