Subcellular Localization And Functional Analysis Of HAOX Protein In Arabidopsis

Photorespiration is a light-dependent process in which molecular oxygen(O₂) is uptake accompanied with release of carbon dioxide(CO₂)from organic compounds.This process requires cooperation of chloroplast,mitochondria,and peroxisome.Photorespiration is catalyzed by ribulose-1,5-diphosphate carboxylase/oxygenase.Ribulose-1,5-diphosphate carboxylase/oxygenase is a bifunctional enzyme,which not only catalyzes the carboxylation of ribulose-1,5-diphosphate with CO₂,but also catalyzes the oxygenation between ribulose-1,5-diphosphate and O₂.Carboxylation results in the formation of phosphoglyceric acid,which enters Calvin cycle and ultimately turn to sugar;and oxygenation results in equimolar amounts of phosphoglyceric acid and phosphoglycolic acid.Phosphoric acid is dephosphorized to glycolic acid,which is toxic to plants.The accumulation of large amounts of glycolic acid can make the color of the tender leaves turn lighter,old leaves turn yellow and so on.Glycolic acid is oxidized to glyoxylic acid by glycolate oxidase,accompanied by the production of H₂O₂.Glyoxylic acid turns to serine and CO₂,while H₂O₂is decomposed by catalase to H₂O and O₂.The degradation of glycolic acid catalyzed by glycolate oxidase is the only known pathway to metabolize glycolic acid in plants.Five genes encoding glycolate oxidase were identified in Arabidopsis genome:GOX1,GOX2,GOX3,HAOX1 and HAOX2.The five glycolate oxidase isozymes are predicted to be located in the peroxisome.GOX1 and GOX2 are highly expressed in the photosynthetic tissues of the plants,and the glycolate oxidase encoded by the two genes participate in photorespiration by using glycolic acid as their substrate.The expression of GOX3 is very low in autotrophic tissue,but is predominent in root and senescent leaves.Though its physiological function in heterotrophic organs is not clear,GOX3 is speculated as a candidate enzyme that mainly metabolizes L-lactic acid in plants.HAOX1 and HAOX2 are mainly expressed in the seeds of different developmental stages,and the two enzymes encoded by them have different properties to the hydroxyl acids in the middle and long chains,indicating that they may involve in the catabolism of fatty acids and proteins.HAOX1 and HAOX2 are focused in this work.At present,there is little research on these two genes,so the functions of them in plants are not very well understood.The transient transformation of onion epidermal cells showed that HAOX1 and HAOX2 are localized in peroxisome.The double mutants of haox1/2were obtained by CRISPR-Cas9 gene editing technique in the previous work of the laboratory.In this paper,we focus on the role of HAOX1 and HAOX2 in the photorespiration pathway and the substrate specificity of HAOX1 and HAOX2.The low CO₂ environment and the condition of diurnal highlight can promote the occurrence of photorespiration.Under the condition of diurnal highlight,cat2mutant,which is lack of CATALASE2,results in oxidative damage to death due to the accumulation of H₂O₂ in vivo,the phenotype of which is called photorespiration phenotype.Blocking of upstream pathway of H₂O₂ production in the cat2 mutant alleviates photorespiration phenotype.It is reported that cat1/2/3 and cat2 share the same photorespiration phenotype,so it can be inferred that the upstream pathway of H₂O₂ production in the cat1/2/3 mutant is blocked,the photorespiration phenotype of the mutant will also be weakened.In this study,we investigated whether HAOX1 and HAOX2 were involved in the photorespiration pathway from the following two aspects:(1)the mutant haox1/2 and Col-0 were cultured in normal and low CO₂ light incubator,If HAOX1 and HAOX2 are functional genes in the photorespiration pathway,then the mutant haox1/2 will lead to the accumulation of glycolic acid because of the lack of function,it can lead to the color of tender leaves becomes lighter,and the old leaves turns yellow and so on.The results showed that there was no significant difference in the growth of haox1/2 and Col-0 under the environment of low CO₂.(2)After the mutants cat1/2/3 haox1/2 were obtained by hybridization,the three mutants of cat1/2/3,haox1/2,cat1/2/3 haox1/2 and Col-0 were cultured in long-day incubator for 10-15 days,and then transferred to the whole day incubator for one week to observe the phenotypes.The results showed that the growth of mutant cat1/2/3 was consistent with that of cat1/2/3 haox1/2,and almost all of them died.The growth of haox1/2 was consistent with that of Col-0,and there was no death phenomenon,which indicated that knockout of HAOX1 and HAOX2 gene in mutant cat1/2/3 could not reduce its photorespiratory phenotype.Through these two experiments,we conclude that HAOX1 and HAOX2 is not a functional gene in the photorespiration pathway.HAOX1 and HAOX2 have different specific affinities to medium-long chain hydroxylic acid substrates.In this paper,leucic,2-hydroxy-4-methylvalerate,2-hydroxyoctanoic acid and 2-hydroxyhexanoic acid were selected to test the substrate tolerance.It was found as the substrate concentration increases,the growth of mutant haox1/2 and Col-0 was stressed,such as root length short and leaves turn white.However,there was no significant difference in the growth status between the two groups.GOX3 had similar substrate specificity with HAOX1 and HAOX2.Therefore,we further verified whether it was caused by GOX3 that the growth of mutant haox1/2 and Col-0 had no significant difference in substrate-specific test.We obtained gox3 haox1/2 mutants by gene editing technique,and then carried out substrate-specific experiments with mutant materials.It was found that when the substrates were glycolic acid,leucic,2-hydroxyhexanoic acid and 2-hydroxyoctanoic acid,both gox3 haox1/2 and Col-0 were stressed,but there was no significant difference in the growth status between the two mutants.These results suggest that although both GOX3 and HAOX1 and HAOX2 are specific to the above substrates,they may not be the key enzymes involved in the metabolism of the above substrates.So even if the three genes were knocked out at the same time,there was no significant difference in growth between the mutant and the wild type.