Gene Isolation And Functional Characterizations Of GmIT1 Regulating Plant Architecture In Soybean

Soybean is one of the important crops in China.The grain contains 40%crude protein and 20%oil,which is much higher than other crops.The protein content is more than twice that of pork and eggs.It has the reputation of "plant meat" and is one of the main sources of protein.Due to the wide application of soybeans in life and industry,China’s demand for soybeans is very high.In recent years,due to China’s low internal self-sufficiency rate of soybeans and insufficient production capacity,China has seriously relied on imported soybeans.China imported soybean 88.511 million tons in 2019,100.33 million tons in 2020 and 96.52 million tons in 2021.The low soybean yield in China is mainly due to two factors:small planting area and low yield per unit area.The unit yield of soybean in China is about 1950 kg/hm2,which is far lower than that of rice and maize.Therefore,the unit yield of soybean still has great potential to improve.Plant architecture improvement is one of the effective means to improve crop yield.By reasonably improving plant architecture,increasing planting density,promoting population light energy utilization and increasing population biomass,and thus increase crop yield.In this study,the mechanism of plant architecture formation and regulation in soybean was studied based on the previously obtained plant architecture mutant.Based on the initial mapping results of ideal Type 1(IT1)mutant and Jidou 12 population,further narrow down the candidate region by expanding the population.The plant type regulation gene GmIT1 was obtained.Cytological observation was conducted on it1 mutants to explore the cytological basis of plant architecture formation.Further analysis of GmIT1 was conducted included bioinformatics,gene expression,subcellular localization,validation of transgenic function,immunofluorescence analysis,suppressor mutant screening and hormone treatment.The regulation mechanism of Gm TI1 was investigated in soybean.The research includes the following aspects:(1)The soybean mutant ideal type 1(it1)displays a pleiotropic phenotype characterized by compact plant architecture,reduced plant height,shortened petioles,wrinkled leaves,and indented seeds.These tissues and organs variation are mainly due to obvious changes in the cell.The epidermal pavement cells of WT plants showed irregular shapes that interlocked with one another via protrusions and indentations,whereas epidermal pavement cells of itl were almost polygonal and protrusions and indentations almost absent.The sizes of mutant pavement cells were also reduced.The direction of microtubules in pavement cells of the WT was randomly distributed.In contrast,microtubules in pavement cells of the itl mutant were well ordered into parallel microtubules.There were more xylem cells in the it1 mutant than in WT plants.Epidermal cells had similar sizes and were arranged neatly,while those of it1 showed irregular dents on the cotyledon surface,and cells in the dents were expanded.(2)A plant architecture related gene GmIT1 was obtained on chromosome 5 by map based cloning using an expanded F2:3 segregation population.There was one mutantion(G-A)was dectect at the 401 base of the CDS sequence in the second extron of candidate gene,resulting in a mutation from glycine in the wild type to aspartic acid on the amino acid sequence.Transgenic experiments have shown that overexpression of Gmitl gene changed plant architecture.Transgenic plants expressing Gmit1 displayed phenotypes similar to that of the it1 mutant:compact and dwarf plant architecture,small and wrinkled leaves,and short petioles Cytological observations of transgenic materials indicate that the epidermal pavement cells of transgenic plants were also polygonal and that protrusions and indentations were almost absent.In cross section,the petiole diameter of transgenic plants was larger than that of WT plants with more xylem cells.(3)The GmIT1 gene encodes α-tubulin,which has three conserved domains,including nucleotide binding site,alpha/beta domain interface and lpha domain interface.The secondary structure of Gmit1 protein changed significantly from β-pleated sheet to Random coil.There are 13α-tubulin genes in soybean.GmIT1 was closely related to Arabidopsis AtTUA3(At5g19770)and AtTUA5(A t5g19780).GmIT1 was expressed in different tissues,with little change expression levels of leaves and grains at different developmental stages.There were many differentially expressed genes involved in xylem development and cell development between wild-type and mutant.GFPtagged GmIT1(GmIT1-GFP)or its mutant in it1(Gmit1-GFP)was localized exclusively in the cytoplasm,suggesting that the single-base substitution did not change its subcellular localization.(4)Microtubule specific depolymerization drug experiments show that the change of microtubule structure directly leads to the change of cell morphology.The GmIT1 gene of these suppressor mutants was sequenced and identified.Suppressor mutants showed varying degrees of plant height recovery.Leaf size,petiole length,and seeds of its mutants were restored to the wild type.The pavement cells of these mutants were also restored to the irregular shape and the microtubules in the pavement cells also restored to random orientation.Five mutants that able to suppress the phenotype of it1 were obtained by EMS mutagenesis in the it1 background.All these mutants contain an additional mutation in the it1 locus.P263S,T191S,P325S,D76V,and G34S repress the it1 phenotype by destabilizing α-α or α-β oligomerization.(5)The mutants and transgenic materials were unable to recover to the level of wild type due to their reduced response to exogenous GA3.Some direct or indirect interactions between microtubules or GmIT1 and the expression of GA and BR-related genes affected the growth and development of plants.Based on the above research results,GmIT1 is a pleiotropic gene that regulates the morphogenesis of soybean plant architecture,and affects many soybean traits.The studies on the it1 and its suppressor mutants provide theoretical and material basis for further study on the molecular mechanism of plant architecture regulation and improvement of soybean plant architecture.