Characterization And Functional Analysis Of Oxalyl-CoA Synthetase In Rice

Oxalic acid is widely distributed in biological systems and known to play functional roles in plants.The gene AAE3 was recently identified to encode an oxalyl-CoA synthetase?OCS?in Arabidopsis that catalyzes the conversion of oxalate and CoA into oxalyl-CoA.It will be even particularly significant to characterize the homologous gene in rice since rice is not only a monocotyledonous model plant,but also a staple food crop.In this study,the molecular,biochemical and enzymatic properties of rice OCS and its biological functions have been investigated by using various approaches,such as bioinformatics,molecular cloning,heterologous expression,biochemical and enzymatic analyses,and also transgenics generation and then analyses on their transcriptome and metabolome.The main results were obtained as described below:1.OsAAE3 encodes an oxalyl-CoA synthetase in rice?OsOCS?A cDNA sequence in the rice genome?Os04g0683700?,homologous to At AAE3,was identified and then amplified by RT-PCR.Only one OsAAE3 gene exists in the rice genome,with 70%amino acid similarity to AtAAE3.The OsAAE3 protein was heterologously expressed in E.coli,then purified by nickel-affinity chromatography.Enzymatic analyses showed that the OsAAE3-encoded protein?OsAAE3?showed activity only with oxalate as its substrate,which specifically catalyzed the conversion of oxalate and CoA into oxaly-CoA.Therefore this encoded protein can be named as oxalyl-CoA synthetase?OCS?.There exist three isozymes for OCS in rice.It was estimated that the molecular weight of holoenzyme is about 110,221,276 kDa,likely consisting of 2,3,4 subunits,respectively.The K_m of the rice OCS for oxalate is 1.72mM,and the optimum pH for its catalysis was shown to be 7.5 in Tris-HCl buffer and the optimum temperature is 38?.The above results established the enzymic fuction of OsOCS and its basic biochemical and catalytic properties.2.Thiols are essential residues of the amino acids in the active site of OsOCSThe active site of OsOCS was also analyzed by using the purified enzyme protein,which was heterologously expressed in E.coli.First,effects of various chemicals on OsOCS activity were investigated.Among more than 19 anion and cation chemicals tested,it was found that Cu²⁺and Zn²⁺inhibited the enzyme activity by more than 90%,whereas no obvious effects were noticed for other chemicals.In addition,two thiol-specific modifying agents,i.e.5'-Dithiobis-?2-nitrobenzoic acid??DTNB?and N-ethylmaleimide?NEM?were used to treat the enzyme?10 min?before determining its activity.The result showed that these two chemicals were able to inhibit the activity of OsOCS by 90%,further supporting that thiol residues are involved in the active site of OsOCS.To confirm the thiol involvement and to further identify which thiols are essential for OsOCS activity,we used site-directed mutagenesis to identify which cysteine is involved in the active site.It was turned out that mutations in either Cys219 or Cys363 of OsOCS resulted in its activity to be almost totally lost,while mutations in Cys287,or 454,or 473 had only partial inhibition on its activity.This result demonstrates that the thiols of Cys219 and 363 act as the essential residues of amino acids in the active site of OsOCS and the others may only play roles in enzyme structure and/or conformation.3.OsOCS is localized to the cytosolThe PSORT and SignalP program indicated a non-organellar localization,both predictions came with low reliability scores.To further investigate the subcellular localization of the OsOCS protein,we expressed green-fluorescent-protein?GFP?fusions in plant cells.Plasmid and binary vectors containing cDNAs encoding both AAE3-GFP and GFP-AAE3 fusion proteins,under control of the strong,constitutive Ubi promoter were constructed.Both AAE3-GFP and GFP-AAE3 fusion proteins were localized to cytosol.4.Generationoftransgenicriceplantswith OsAAE3 interfered and overexpressedThe interference and over-expression sequences of OsAAE3 were cloned using reverse transcription-polymerase chain reaction?RT-PCR?with specific primers designed on the basis of the registered rice cDNA sequence.Each fragment was then ligated into an constitutive expression vector named pYL and pOX.The constructed vectors were then transformed into rice callus,respectively,by Agrobacterium mediated infection.The positive transgenic plants were selected by PCR and hygromycin.Through 3 generations of multiplying and selection,homozygous offspring with OsAAE3 down-regulated and OCS activity significantly decreased were acquired,but no ideal transgenic lines were obtained for over-expression,likely due to occurrence of co-suppression.The down-regulated transgenic lines provide basic materials for further functional analyses of OCS in rice.5.OCS is involved in oxalate metabolism and growth/development of riceOxalate measurement revealed that,when OsAAE3 was down-regulated and OCS activity was decreased in the transgenic plants,total oxalate content was increased by around 50%and the soluble content was increased by 85%.These transgenic plants also showed significant phenotypes,typically with²5%decrease in their kernel number per spike and 4 days earlier for their flowering time.6.Role of OCS in stress resistanceOxalate has been demonstrated to be involved in various biotic/abiotic stresses,such as metal toxicity,pathogen and insect attack.We tested how the transgenic rice plants with AAE3 down-regulated and oxalate increased respond to Al toxicity.In comparison with WT,the RNAi plants displayed a slower growth rate of roots than WT in the absence of Al,but a significantly higher root elongation rates in the presence of Al,indicating increase resistance to Al stress when OsAAE3 is suppressed.We further tested responses of the OsAAE3 suppressed transgenic rice plants to bacterial blight.The results demonstrated that the transgenic plants with OsOCS down-regulated and oxalate increased were more susceptible to bacterial blight as compared to WT.The above results indicate that OCS may play totally distinct roles in different stress responses.7.Transcriptome and metabolome changes in response to OCS suppressionRNA-Seq analysis showed that:?i?totally 80 genes were significantly regulated by suppressing OsOCS,among which 59 up-regulated and 21 down-regulated;?ii?those genes that are involved in transport and metabolism of substances are altered with highest frequency,and 26 genes are highly regulated.Of them those that are involved in nitrogen assimilation and redox homeostasis were down-regulated by more than 50%,such as SOD,nitrite reductase,etc,while succinic dehydrogenase was up-regulated by 100%,which is known to be involved in energy metabolism.GC-MS analysis found that glucoce and fructose were reduced by about 35%in the RNAi transgenic lines,succinic acids by 25%,malate by 15%,and phenylalanine by 36%.As described above,by molecular,biochemical and enzymatic analyses we first demonstrate that OsAAE3 encodes an oxalyl-CoA synthetase in rice,which can very specifically catalyze the conversion of oxalate and CoA into oxlaly-CoA.In addition,it was revealed that thiol residues are essential amino acid groups in OCS active site and its activity may likely be regulated by redox state in plants.Further functional analysis shows that OCS is involved in oxalate metabolism and associated with flowering time and stress resistance,thus logically speaking,these processes might also be controlled by redox homeostasis in plants.