| The cell wall is a unique cell structure enclosing plant cells,which determines the direction of cell growth during plant growth and development,and protects cells against various external environmental stresses.The plant cell wall is a complex network,primarily composed of polysaccharides,which also presents the largest sink for photosynthetically fixed carbon.The biosynthesis of plant cell walls is a highly complex and dynamically regulated process.In order to adapt to the external environment,the plant cells require flexible dynamic regulation to ensure the rapid cell growth and resistance to mechanical stresses while maintaining the cell wall integrity.This process requires the concrete actions of a series of proteins such as multiple enzymes and metabolic intermediates,intracellular proteins and the transporters of cell wall precursors.It is a promising way to genetically modify key genes involved in cell wall biosynthesis and regulation for improving agronomic traits and stress resistance.Therefore,it is of great theoretical significance and application value to identify cell wall genes with good potential for genetic improvement of crops.Rice is one of the important food crops in China.In recent years,soil salinization in China has been becoming serious due to the accelerated industrialization process,climate change,and unreasonable irrigation.Salt stress is one of the abiotic stresses restricting rice production.Under salt stress conditions,rice is negatively affected by multiple factors such as ion toxicity and osmotic stress,resulting in impaired plant growth and development.The cell wall is the first physical barrier against abiotic stress and is the organ of rice to perceive salt stress.Therefore,The physicochemical properties of cell wall directly determine the tolerance of rice to salt stress.Many previous studieson cell walls have revealed that cell wall polysaccharides play important and wide roles in plant resistance to various stresses.However,the synthases of substrates required for cell wall polysaccharide polymerization and their responses to abiotic stresses remain largely unknown.UGEs,encoding UDP-Gal/Glc epimerases,catalyses the bioconvertion of UDP-Gal and UDP-Glc,which provides the substrate sugar donors for the biosynthesis of cell wall polysaccharides,glycoproteins,and glycolipids.In this study,we studied the biological functions of Os UGEs family genes in rice.Based on the information of the previously characterized UGE2,we generated the UGEs overexpressing transgenic plants and knock-out lines by the CRISPR/Cas9 approach,and identify the effects of UGEs on cell wall polysaccharides metabolism,agronomic traits,and salt stress resistance.The main results ara as follows:(1)The knock-out transgenic lines of uge1,uge3 and uge4 were constructed by the CRISPR/Cas9 gene editing technology,and homozygous lines were selected out by the PCR analysis.Among those transgenic lines,the uge3 mutant showed serverely stunted plant growth,while both uge1 and uge4 mutants showed indistinguishable phenotypes as compared with the wild-type(WT)plants.By contrast,OsUGE3 overexpression lines(OsUGE3-Ox)displayed the opposite phenotypes.(2)Phenotypic analyses showed that the OsUGE3-Ox plants showed significant increases in plant height,dry biomass,dry spike,1000-grain weight as compared with wild type plants.Notably,the overexpression of UGE3 led to a substantial improvement in stem mechanical strength,including breaking force,extension force,and puncture force,while the loss-of-function of UGE3 resulted in a reduction in mechanical strength.(3)Expression analysis showed that OsUGE3 was broadly expressed in various tissues and organs of rice.The enzymatic activity analysis revealed that the UGE3 could catalyze the bioconversion of UDP-glucose and UDP-galactose.(4)The cell wall structure and composition analyses showed that OsUGE3 increased the accumulation of cell wall polysaccharides(cellulose and hemicellulose)and soluble sugars by supplying the nucleotide sugar substrates,which results in a reinforced cell wall and inceased biomass.The expression of OsUGE3 could be upregulated by the salt stress.The OsUGE3 overexpression lines showed significantly increased salt tolerance,while the knock-out lines were super sensitive to salinity.To determine whether high salinity impacts on growth of OsUGE3 transgenic plants as a consequence of ion toxicity or osmotic stress,we treated rice seedlings at the threeleaf stage with mannitol,which can induce osmotic stress but not ion toxicity.The results showed that the OsUGE3-OX plants had significantly increased fresh weight and survival rate compared with wild-type plants,whereas the uge3 mutants displayed increased sensitivity to osmotic stress imposed by mannitol.(5)OsUGE3 plays a key role in maintaining the balance of Na+/K+ ratio in seedlings under salt stress condition.OsUGE3 overexpressed may maintain the Na+/K+ ratio in plants to enhance their salt tolerance by affecting the expression of genes related to K+ and Na+ transport.(6)Analysis of cell wall components showed that the overexpression of OsUGE3 gene increased accumulations of cellulose,and hemicellulose to enhance the salt tolerance of plants.(7)Combined with the study results,we proposed a hypothetical model that highlights how OsUGE3 positively regulates cell wall polysaccharides biosynthesis for biomass production,mechanical strength,and salt stress tolerance.The OsUGE3 catalyzes the bioconversion between UDP-Gal and UDP-Glc,which provides sugar substrates for the biosynthesis of cell wall polysaccharides and soluble sugar and strengthens cell wall structure in rice.The reinforced cell walls and increased accumulations of cellulose,hemicelluloses,and soluble sugars resulted from the overexpression of OsUGE3 improve biomass production,mechanical strength,and salt stress tolerance in rice.Hence,OsUGE3 is identified to be a promising targeted gene. |
