The Role Of SlOCD And SlAAE In Oxalate Degradation And Their Impacts On Transcriptome And Metablome Of Tomato Fruit

Oxalic acid is the simplest dicarboxylic acid,which exists ubiquitously in algae,fungi,lichens,plants and animals.Due to its high acidity（pKa1=1.25 and pKa2=4.27）,reducibility,and chelation ability,oxalic acid plays an important role in many biological and metabolic processes in plants.For instance,oxalic acid has been reported to be involved in the regulation of intracellular p H,inorganic ions homeostasis,and redox status,chelation and detoxification of metals,and plant defense.Besides,excessive accumulation of oxalic acid results in metabolism disorder,increase in the susceptibility to pathogens,and reduction in mineral nutrition quality.Therefore,the content of oxalic acid in plants has to be precisely controlled through the synthesis and catabolism process to maintain the normal cellular metabolism.The biological function of oxalic acid has been studied comprehensively,but as an anti nutritional factor,its effect on fruit quality and its underlying mechanism are still unclear.Due to the lack of oxalate oxidase activity,oxalate acetylation catabolic pathway is the most important oxalate degradation pathway in dicotyledons.Although it is confirmed that oxalate acetylation metabolic pathway is catalyzed by four enzymes successively（Acyl activating enzyme3,AAE3;Oxalyl-CoA decarboxylase,OCD;Formyl-CoA hydrolase,FCH;Formate dehydrogenase,FDH）,this pathway has not been fully studied yet.Therefore,oxalate acetylation metabolism and its effects on fruit quality deserve further study.Tomato is a model plant for studying fruit development,and oxalic acid is an anti nutritional factor that affects the quality of fruit.Therefore,study on oxalate acetylation metabolism of tomato and its effect on fruit quality will not only help to understand the regulation mechanism of oxalate metabolism,but also provide an important theoretical basis for improving plant stress resistance and food quality through regulating oxalate metabolism.The main results are as follows:1.Oxalic acid is activated in the cytoplasm and decarboxylated in the endoplasmic reticulumThe oxalic acid biodegradation pathway is still controversial.By constructing the fusion protein of green fluorescent protein（GFP）and SlAAE3 protein,we proved that SlAAE3 functions in the cytoplasm when transiently transformed into tobacco leaves.Similarly,SlOCD protein was also fused with GFP,then introduced into tobacco leaves with various organelle markers.The colocalization indicated that SlOCD protein was localized in the endoplasmic reticulum.It can be concluded that oxalic acid is catalysed by the SlAAE3 protein to form oxalyl coenzyme A,which occurs in the cytoplasm.Then oxalyl coenzyme A is transported to the endoplasmic reticulum,where decarboxylation is carried out under the catalysis of SlOCD protein.2.SlOCD may play an important role in embryo developmentIn order to explore the biological function of OCD,we first identified the enzymatic characteristics of SlOCD.Due to the limited detection capability and the extremely similar polarity between oxalyl-CoA and formyl-CoA,it is very difficult to isolate and identify the products.Therfore,we failed to detect the degradation of oxalyl CoA by SlOCD in vitro through biochemical methods.Then,we tried to construct a Slocd knockout line in Micro-Tom tomato background.However,we failed to obtain any homozygote after several generations.We believe that SlOCD may play an indispensable role in the process of embryonic development.3.SlAAE3 can catalyze the degradation of oxalic acidOur previous research showed that there are two SlAAE3 proteins in tomato,namely SlAAE3-1 and SlAAE3-2.Through prokaryotic expression system,we purified SlAAE3-1 and SlAAE3-2 proteins,and successfully proved that they can catalyze oxalic acid degradation.SlAAE3-1 showed a high enzymatic activity,with K_m=223.8±20.02μM and V_max=7.908±0.606μMol mg^-1protein min^-1,whereas the degradation rate of oxalic acid by SlAAE3-2 is too low to detect.Further we constructed Slaae3-1 knockout mutant in Micro-Tom background,and carried out oxalic acid feeding experiment with detached pinnate compound leaves.When feeding with oxalic acid,the mutant material displayed more severe leaf tissue necrosis compared with the wild type.These results proved that SlAAE3 has the function of degrading oxalic acid both in vitro and in vivo.4.The absence of SlAAE3 improves fruit quality of tomatoWe measured the content of main sugars and organic acids in the fruits of Slaae3-1 knockout lines and wild type,respectively.Compared with the wild type,the sugars content（glucose,fructose and sucrose）in the mutant had been significantly increased,while the content of organic acids（citric acid and malic acid）had been significantly reduced,but the content of oxalic acid increased slightly in the mutant lines.Finally,the sugar-acid ratio in the mutant fruits is twice-fold higher than wild type.In addition,the contents of major mineral elements（Mg,Zn,Fe,Ca,P and Mu）in fruits of knockout lines at mature stage stage are also higher than wild type.Therefore,the absence of SlAAE3-1 improves the quality of tomato fruit.5.SlAAE3 involves in the redox metabolism of tomato fruitIn order to further clarify the regulatory mechanism of SlAAE3-1 on tomato fruit quality,we constructed SlAAE3-1 overexpression lines.Transcriptomic and metabonomic analyses were conducted on fruits of SlAAE3-1 overexpression line and wide type material at different maturity stages and principal component analysis（PCA）was conducted on the data.The results showed the overexpression of SlAAE3-1 had an impact on the transcription and metabolism of tomato fruits.GO analysis of differentially expressed genes in transcriptome at various stages（20 days post-anthesis,20DPA;mature green,MG;pink,Pk;red rip,RR）showed that they were mainly enriched in the redox pathway.At the same time,a total of 21metabolites were identified with a fold change more than 10 times in the metabolome data,and 14 of them（up to 66.7%）belong to phenolic acids which are thought to be reducible.It can be concluded that SlAAE3 involved in the redox metabolism of fruit.In summary,acetylation pathway plays a role in the degradation of oxalic acid in tomato,and this pathway can affect the metabolism of major sugars and organic acids in tomato fruits through the process of redox metabolism,thereby affecting the quality of tomato fruits.