Functional Study Of Glutamate-rich Proteins PtrGARP1;1 And PtrGARP1;2 In Stem Growth Of Populus Trichocarpa

Wood is not only an important raw material for the national economy,but also plays an important ecological function in the dual carbon goals.Wood is the product of lateral growth of forest stems,and its development undergoes a series of biological processes that are strictly genetically regulated,including:vascular cambium formation,division and differentiation,secondary wall thickening,and programmed cell death.Currently,a variety of plant hormone molecules,transcription factors,as well as small peptides,small RNAs,and kinases have been identified to be involved in wood formation.However,the molecular basis of genetics in forest stem growth is still relatively weak.Identification and characterization of important proteins or key genes involved in the growth of forest stems can help to resolve the molecular mechanism of wood formation,and is also of great value for the molecular breeding of wood traits.In this study,we applied proteomic strategies to identify proteins related to woody growth of young stems of Populus trichocarpa,and used genetics and wood microstructure analysis to functionally investigate the genes encoding glutamic acid protein（GARP）and obtained the following main results:Using differential proteomics,we carried out the excavation of proteins related to lignified growth of young stems of Populus trichocarpa.By mass spectrometry and Mascot identification,165 differential proteins in lignified growth of young stems were identified,and these protein belonged to 14 functional categories,including carbon metabolism,protein degradation,redox,cell wall synthesis and modification,signal transduction,and amino acid metabolism and so on,among which the total number of proteins in the first four functional categories accounted for57.6%,indicating that they were the main biological processes for the lignified growth of young stems of Populus trichocarpa.In addition,the proteins with consistently increasing abundance in the 1st,3rd,and 5th internodes accounted for about 38.2%,suggesting that these proteins may be involved in the lignified growth of young stems.Among them,protein of unknown function No.50,a glutamate-rich protein named Ptr GARP1 strongly accumulated in abundance with increasing lignification of young stems,suggesting that it may be an important genetic factor for wood formation.The genomic search of Populus trichocarpa revealed that there are two genes encoding Ptr GARP1,named Ptr GARP1;1 and Ptr GARP1;2.Sequence searches indicated that Ptr GARP1homologs existed in plants（mainly woody plants）with the number of amino acids varying from125 to 256,and the amino acid identity ranging from 32%to 99%.The results of fusing green fluorescent protein GFP and transforming poplar leaf protoplasts showed that Ptr GARP1;1-GFP and Ptr GARP1;2-GFP may be localized in the cytoplasm.Driving GUS reporter gene expression indicated that the activities of the promoters of Ptr GARP1;1 and Ptr GARP1;2 were mainly present in the lignified vascular tissues of Populus trichocarpa,such as fibers and vessels in stem and root xylem,implying that Ptr GARP1;1 and Ptr GARP1;2 may be involved in wood formation.Based on the Cas9/g RNA editing technology,single-gene and double-gene allelic editing mutant lines of the Ptr GARP1;1 and Ptr GARP1;2 were created for 4,5 and 5 respectively.Compared with wild type（WT）of Populus trichocarpa,the ptrgarp1;1 and ptrgarp1;2 mutants had no significant differences in plant height and diameter;however,the ptrgarp1;1/1;2 double mutants showed a significant reduction in plant height,diameter,the length and the number of the internodes,indicating that knockout of Ptr GARP1;1 and Ptr GARP1;2 suppressed the growth of stems of Populus trichocarpa,and the Ptr GARP1;1 and Ptr GARP1;2 were functionally redundant in stem growth.The microscopic observation of the cross section of the main stem of young trees showed that compared with WT,the ptrgarp1;1/1;2 double mutant had weakened division activity of cambium,decreased the area of secondary xylem,and increased the thickness of the secondary cell wall of mature xylem fibers;the ptrgarp1;1 and ptrgarp1;2 single mutants were not significantly different in these respects.In addition,the ptrgarp1;1/1;2 double mutants had a significant increase in the proportion of long fibers and vessels in the secondary xylem,as well as the proportion of larger fibers and vessels in the horizontal axis,indicating that the Ptr GARP1;1 and Ptr GARP1;2 gene knockout increased the size of wood fibers and vessels.The assay showed that the cellulose and lignin contents of the ptrgarp1;1/1;2 double mutants were slightly increased compared with WT.Genetically transformed Populus trichocarpa and created Ptr GARP1;1 and Ptr GARP1;2overexpression materials.The transcriptional levels showed that 31 and 23 transgenic plants overexpressing Ptr GARP1;1 and Ptr GARP1;2 respectively.Phenotypic observation showed that compared with WT,plants overexpressing Ptr GARP1;1 and Ptr GARP1;2 showed a slight increase in plant height,diameter,and the number of internodes,but no significant changes in the length of internodes.The microscopic observation of the cross section of the main stems showed that the fiber cell walls of the mature secondary xylem in Ptr GARP1;1 and Ptr GARP1;2overexpression were significantly thinner,indicating that overexpression of this genes reduced the thickness of the secondary walls of wood in Populus trichocarpa.A yeast library for gene expressing in young stems and lignified stems of Populus trichocarpa was constructed,and the number of library cells reached 4.68×10 ⁷cfu/ml.Using Ptr GARP1;1 as the bait protein,the yeast expression library was screened,and 6 candidate interacting proteins were obtained:COP9 complex protein subunit,cytochrome P450,Ca²⁺binding protein,ribosomal protein small subunit S25e;4 candidate interacting proteins were screened for the Ptr GARP1;2:leucine-rich protein kinase,protein of unknown function,trichome birefringence family protein and ribosomal protein small subunit S25e.The ribosomal protein small subunit S25e is a common candidate protein for the Ptr GARP1;1 and Ptr GARP1;2.It will be our next breakthrough to analyze the mechanism of Ptr GARP1 regulating stem growth.