| Peroxisomes are multifunctional organelles in plant cells responsible for key metabolic processes such as fatty acid degradation,hormone synthesis,photorespiration,and reactive oxygen species detoxification,and play an important role in plant growth,development,yield and stress response.Therefore,peroxisome-related studies have high scientific significance and agricultural application value.Peroxisomes have complex metabolic network with high specificity on tissue and species.Profiling of peroxisomal protein composition is a prerequisite for the comprehensive understanding of their metabolic function.Proteomic analysis based on organelle isolation,purification,and mass spectrometry is the main tool for systematically identifying peroxisomal proteins despite of its severe limitation by the tissue specificity of peroxisomal protein composition.But by direct prediction of peroxisomal proteins through signal peptides,the limitation of tissue specificity in gene expression can be effectively overcome.Therefore,revealing the localization mechanism of peroxisomal proteins and identifying more peroxisomal proteins can help to comprehensively resolve their protein composition and metabolic network.In addition,previous studies have conducted a series of systematic identification of peroxisomal proteins in dicotyledonous plants such as Arabidopsis,soybeans,and spinach.However,in rice,the model plant of the monocotyledonous grass family,the systematic study of peroxisomal proteins has been studied relatively little.Limited by species specificity,there are numerous unknowns regarding in the the protein composition and physiological function of rice peroxisomes.In this work,a systematic identification of potential plant peroxisomal proteins was firstly carried out based on mass spectrometry and bioinformatics methods,combined with subcellular localization experiments.On the one hand,the author successfully isolated and purified peroxisomes from Arabidopsis thaliana green leaves to carry out protein profiling.On this basis,experiments were carried out to validate 9peroxisomal proteins,including At HRLP（HEAT Repeat Like Protein）,At GGH2（γ-glutamyl hydrolase 2）,At ALDH11A3.4（Aldehyde dehydrogenase 11 A3 isoform 4）,At SDRf（Short-chain Dehydrogenase/Reductase f）,At SDRg,At SDRi,At SDRj,At AAC1（ADP/ATP carrier 1）,At OEP16-3（Outer chloroplast Envelope Protein of 16k Da-3）.Meanwhile,7 rice peroxisomal proteins,including Os HRLP,Os OEP16-3L1（OEP16-3 Like 1）,Os OEP16-3L3,Os OEP16-3L4,Os FIS1A（Fission 1A）,Os FIS1B and Os FIS1C,were identified as they were homologous of Arabidopsis peroxisomal novel proteins.Among them,the membrane protein families such as OEP16-3,AAC and FIS1 were localized not only in peroxisomes,but also in organelles with closely related metabolic functions with peroxisomes such as mitochondria and chloroplasts,implying that the functions of these proteins are related to trans-organelle transport and organelle interactions.On the other hand,in this work,the author also carried out bioinformatic analyses on the rice proteome to identify and validate other new peroxisomal proteins containing PTS1.A total of 25 nevel rice peroxisomal proteins were found which belong to Os AAE（Acyl-Activating Enzyme）family,Os ATFP（Acyl-Transferase Family Protein）family,Os SDR（Short-chain Dehydrogenase/Reductase）108E subfamily,Os GST（Glutathione S-transferase）family,Os HSP（Heat Shock Proteins）family and Os ND（type II NAD（P）H dehydrogenases）family.The identification of these novel proteins has substantially broadened the comprehension of rice peroxisomal metabolic network and protein composition of peroxisomal proteome.The work also examined the specific subcellular localization strategies of the At HRLP and Os HRLP proteins,which were identified in peroxisomal proteome.The author found that HRLP is a conserved and unique peroxisomal protein in all higher embryonic plants,including mosses,and that HRLP contains a large number of paired helices,which is similar to the Heat Repeat domain responsible for protein-protein interactions,and is therefore named the HEAT Repeat Like Protein which indicates its functional importance.The absence of At HRLP resulted in embroy lethality in Arabidopsis during embryonic development,indicating its significant function.Additionally,whilst the C-terminus of HRLP includes a peroxisomal signaling peptide,PTS1,which was both conserved and effective,the absence of PTS1 did not impact its peroxisomal localization.Furthermore,HRLP lacked PTS2（Peroxisomal Targeting Signal type 2）,suggesting the existence of other targeting signals in addition to PTS1and PTS2 in HRLP.To demonstrate this,the author utilized a Saccharomyces cerevisiae system to create deletion mutants of yeast PTS1 and PTS2 receptors Sc PEX5（Peroxin5）and Sc PEX7（Peroxin 7）.It was found that At HRLP can still localized to peroxisomes in these yeast mutants,fully demonstrating that HRLP has a special localization mechanism independent of known PTS1 and PTS2 receptors.Finally,this work explored the physiological role of Os HRLP in rice.The author utilized CRISPR/Cas9 to create rice mutants of Os HRLP.The results revealed that,unlike the oilseeds of Arabidopsis,the typical starch seeds of rice were not substantially impacted by HRLP deficiency.Furthermore,the seed development,germination,and seedling morphogenesis of the Oshrlp mutants were normal,illustrating that HRLP is not necessary for starch seed development and germination.The Os HRLP gene complemented the embryonic lethal phenotype of the Athrlp Arabidopsis mutant.This suggests that At HRLP and Os HRLP have conserved biochemical functions even though the physiological functions in the development of oily and starch seeds are significantly different.The authors further revealed that Os HRLP has an important role in rice heat stress response and is essential for maintaining normal growth and photosynthesis in rice under high temperature.Under heat stress,the rice Oshrlp mutant showed yellowing of leaf color,its chlorophyll content,and net photosynthetic rate were significantly lower than those of the wild type,its intercellular CO2 concentration was higher,and there was almost no accumulation of starch grains in its chloroplasts,suggesting that photosynthesis was significantly inhibited as well as that the Os HRLP protein and peroxisomes have important roles in rice photosynthesis.Meanwhile,antioxidant enzyme activities were elevated and the lipid peroxidation product malondialdehyde was significantly accumulated in the rice Oshrlp mutant,suggesting that there was an abnormality in its reactive oxygen metabolism.Furthermore,a transcriptome analysis was carried out on the Oshrlp mutants and wild type Zhonghua 11 of rice subjected to heat stress.Genes related to metabolic pathways including detoxification of reactive oxygen species,β-oxidation,glyoxylate cycle,and photorespiration showed significant upregulation in Oshrlp,while there were abnormalities in the expression of genes associated with chlorophyll synthesis and photosynthesis.Multiple genes associated with the metabolism of carbon and amino acids were significantly upregulated in Oshrlp,providing further evidence of the significance of Os HRLP and peroxisomes for rice growth and photosynthesis during exposure to heat stress.In summary,this work used protein mass spectrometry and bioinformatics analysis to systematically analyse the plant peroxisomal proteome and extracted a large number of novel peroxisomal proteins.In addition,a systematic analysis of the rice peroxisome proteome was performed,which has been relatively lacking in similar studies before,providing rich genetic resources for understanding the function of peroxisomes in plant in crop production and development.In this work,the novel peroxisomal protein HRLP,which is specific and conserved in higher plants,was also analysed in detail.It was found that HRLP has a unique novel,peroxisomal localization mechanism and plays an important role in the development of oilseeds and the growth and photosynthesis of rice under high temperature.This not only provides a theoretical basis for the study of crop peroxisomal proteins,but also provides new research directions and important genetic resources for improving the quality of oil crop seeds and enhancing crop stress resistance in agricultural production. |
