Study On The Function Of Populus Trichocarpa GDPD-LIKE Genes And Its Coding Protein In Vitro Enzyme Activity

Plant cell plasma membrane is an important structure to separate the cytoplasm and apoplast,protect cells,exchange nutrient molecules and transmit signals.The membrane lipids biosynthesis,circulation and transportation,and dynamic modification play important roles in cell function.Glycerophosphodiester phosphodiesterase（GDPD）catalyzes the hydrolysis of glycerophosphatediester into glycerol-3-phosphate and corresponding alcohols.GDPDs widely exist in animals,plants and bacteria and involve in the phospholipid degradation pathway.Interestingly,higher plants（including trees）have evolved a unique GDPD-LIKE family,but no GDPD activity.It is located in the cell membrane,which may be related to the function of plant cell wall.Atpresent,little is known about the genetic function and biochemical enzyme activity of GDPD-LIKE small family genes.This study took Populus trichocarpa as the main research object,and identified the functions of the two kinds of genes of the GDPD-LIKE subfamily of Populus trichocarpa by using genome editing technology,growth phenotype analysis,quantitative lipidomics,ultrastructural observation,and protein biochemical characteristics analysis,and preliminary explored the in vitro enzyme activity of GDPD-LIKE protein.The main research results are as follows:The BLAST of Populus trichocarpa genome data showed that there were only four GDPD-LIKE genes（PtGDPDL1-4）,and two GDPD-LIKE domains were visible in the amino acid sequence of each gene,which were GPI anchored proteins located in the plasma membrane.According to 46 evolutionary trees of GDPD-LIKEs of 13 species,they are divided into three clades:Ⅰ,Ⅱ and Ⅲ.GDPD-LIKEs of clade Ⅰ and Ⅱ are unique to seed plants,and GDPD-LIKEs of clade Ⅲ are unique to spore plants.Plant tissue transcriptome data showed that clade I GDPD-LIKEs were widely expressed in plant vegetative organs,and class Ⅱ GDPD-LIKEs were specifically expressed in male flower organs.RT-PCR analysis showed that PtGDPDL1 and PtGDPDL2 were expressed in the vegetative organs of Populus trichocarpa,belonging to clade I GDPD-LIKEs,while PtGDPDL3 and PtGDPDL4 were specifically expressed in male flower organs,belonging to clade Ⅱ GDPD-LIKEs.Based on Cas9/gRNA genome editing technology,Populus trichocarpa PtGDPDL1 and PtGDPDL2 gene single and double mutant plants were created.Compared with the wild type（WT）,the leaves of the ptgl1gl2 double mutants lost green and the chlorophyll content decreased.In addition,it was found that ptgl1gl2 plants would have severe premature senescence when cultured in the medium,and the expression level of senescence related gene PtLKR was increased about 7 times,and there were no significant correlation between premature senescence of ptgl1gl2 plants and nutrient deficiency.Interestingly,the growth of ptgl1gl2 plants will lead to a rapid decline in the pH value of the culture medium.Adding plasma membrane H⁺-ATPase inhibitor to the culture medium can significantly rescue the chlorosis defect of ptgl1gl2 leaves.Pot culture of ptgl1gl2 plants rescued the premature senescence phenotype,suggesting that the premature senescence was caused by pH imbalance of cell membrane.However,there was no significant difference between ptgl1 and ptgl2 single mutants,indicating that PtGDPDL1 and PtGDPDL2 genes were functionally redundant.After pot culture in greenhouse for 3 months,the plant of ptgl1gl2 was significantly shorter than WT,the leaf was small and thin,the mesophyll cells were small,the groove shape of leaf epidermis cells was weakened,the morphology of stomatal guard cells was deformed and the chloroplast was less,and the water loss rate of leaves was significantly increased;The main root is short,the number of lateral roots is large and long,and the root hairs are seriously damaged.In addition,the stem of ptgl1gl2 plant was significantly thinner,the thickness of phloem tissue was unchanged,but the primary phloem cells were swollen and deformed,the secondary phloem range was smaller,and the number of cells was less;the secondary xylem area was also significantly thinner,with fewer cell layers,smaller fiber and vessel cells and thinner cell walls.The fiber and vessel cells of ptgl1gl2 plants were difficult to be stained by acid fuchsin,and the content of wood cellulose decreased significantly,while the content of lignin increased;immunofluorescence assay of cell wall polysaccharide also showed that the content of cellulose in cell wall of stem tissue decreased,the content of galactose in phloem cell wall decreased significantly,the content of xylan in xylem and secondary phloem cell wall increased,and the content of xylan and mannan in phloem cell wall decreased.Clade Ⅱ GDPD-LIKE genes of Populus trichocarpa is specifically expressed in male inflorescences,indicating that it may play a role in reproductive function.However,it is difficult to obtain genetic evidence for poplar because of its long reproductive cycle.The genetic system of Arabidopsis thaliana was used to identify the function of PtGDPDL3/4.Its orthologous gene in Arabidopsis is AtGDPDL6/7.Analysis of gene transcription and promoter tissue activity showed that AtGDPDL6/7 was specifically expressed in mature pollen grains and pollen tubes of Arabidopsis.The T-DNA single mutants gl6-1,gl6-2,gl7-1 and gl7-2 had no phenotypic difference with WT,and the AtGDPDL6/7 double mutant gl6-1;gl7-c1 and gl6-c1;gl7-1 was created under the single mutant background by genome editing technology,the double mutants had no obvious nutritional growth defects,but had severe sterility.The results of reciprocal cross experiment showed that the AtGDPDL6/7 double mutant was male gametophytic sterile.Pollen germination experiments in vivo and in vitro showed that the double mutant pollen broke immediately after germination.The results of electron microscopic observation showed that the pollen tube wall of AtGDPDL6/7 double mutant was significantly thinner.Immunohistochemistry of cell wall polysaccharide showed that the cellulose content in the cell wall of pollen tube of the double mutant was significantly reduced,while the accumulation of pectin and callose had no significant change.It was further found that under the background of double mutant,the polarity of cellulose like synthase AtCSLD1/4was abnormal at the top of pollen tube.Expression of Populus trichocarpa PtGDPDL3/4 gene to AtGDPDL6/7 double mutant completely restored the development defect and sterility of pollen tube of AtGDPDL6/7 double mutant,indicating that PtGDPDL3/4 was involved in the deposition of cellulose in pollen tube cell wall and reproductive function.To judge the enzyme substrate of GDPD-LIKE protein,the T-DNA insertion mutant of AtGDPDL3 gene was identified,and the mutant showed root hair development defect.Quantitative lipomics analysis of mutants showed that there was no difference in the level of gl3-1 mutant phospholipids,but the level of glycosylinositol phosphateceramide（GIPC）,the main component of sphingolipid,was significantly increased.The gl3-1 mutant was treated with various intermediate molecules in the GIPC metabolic pathway.The results showed that ceramide（Cer）molecules could significantly rescue the root hair development defects of the mutant.It was speculated thatGDPD-LIKE protein had GIPC-PLC activity.The FLAG-GDPDL3 and its point mutation genotypes GL3^D86A,GL3^D128A,GL3^H289A were used to rescue the gl3-1 mutant phenotype.It was found that GL3^H289A gene had the worst effect on the rescue of root hair of the mutant,so GL3^H289A was the mutation of the key active site of GDPDL3.The FLAG-GDPDL3 and FLAG-GDPDL3^H289A active proteins expressed in Arabidopsis thaliana were purified based on FLAG tag affinity.The enzyme activity in vitro was analyzed using Arabidopsis GIPC as substrate.The results showed that FLAG-GDPDL3 protein had significant GIPC hydrolase activity,while the negative control protein FLAG-GDPDL3^H289Acould hardly detect GIPC hydrolase activity.