Functional Analysis Of OsACL5,a Gene Putatively Encoding Thermospermine Synthase

Rice direct seeding is a simple and labor-saving cultivation method,which alleviates the seasonal lack of agricultural labor force in China.However,there are several problems in the production of direct seeding rice,such as the low emergence rate and irregular emergence.These problems may induce the loss of grain yield.The length of mesocotyl and coleoptile has large effects on rice seedling emergence.Identification of the genes for mesocotyl and coleoptile elongation provide useful information for the development of rice varieties suitable for direct seeding.In this study,the function of OsACL5,a gene putatively encoding thermospermine synthase in rice,was preliminary analyzed.We found that OsA CL5 negatively regulates grain weight and grain yield per plant.Moreover,OsACL5 has negative roles on the length of mesocotyl and coleoptile,and subsequently affects the emergence rate of direct seeding.These data provided important gene resource for developing rice varieties suitable for direct seeding.The major results are as follows:1.qPCR and GUS staining indicated that OsACL5 was constitutively expressed in all tested tissues,with the highest expression levels in the nodes and the lowest expression levels in leaves.The expression level of OsACL5 increased with panicle elongation,but it declined significantly after reaching the summit.2.To investigate the subcellular localization of the OsACL5 protein,the coding region of OsACL5 was fused to GFP to generate a fusion construct.OsACL5-GFP signal showed that OsACL5 was mainly located in the nucleus and cytoplasm.3.To uncover the function of OsACL5 in rice,we generated CRISPR/Cas9 and overexpression constructs of OsACL5,which were then transformed into rice variety Nipponbare.Two CRISPR/Cas9 knockout mutants（osacl5-1 and osacl5-2）and two overexpression lines（OsACL5-OE-1 and OsACL5-OE-2）were selected and used for further investigation.Phenotypic analysis indicated that knockout of OsACL5 promoted internode elongation and increased plant height.Overexpression of OsACL5 reduced plant height.In addition,OsACL5 had negative effects on the diameter of stem.4.The 1000-grain weight in the knockout mutants of OsACL5 was significantly higher than that in wild-type.Conversely,the 1000-grain weight of overexpression lines was obviously reduced.These data showed that OsACL5 decreased rice grain weight.Scanning electronic microscopy analysis showed that OsACL5 negatively control grain size through regulating cell size of rice glume.5.In 2020,the grain yields per plant of osacl5-1 and osacl5-2 mutants were 21.9%and 19.7%higher than those of wild-type,respectively.Conversely,the grain yields per plant of OsACL5-OE-1 and OsACL5-OE-2 were decreased by 22.2%and 41.2%,respectively.In 2021,the grain yields per plant of osacl5-1 and osacl5-2 mutants were 22.2%and 16.4%higher than those of wild-type,respectively.Conversely,the grain yields per plant of OsACL5OE-1 and OsACL5-OE-2 were decreased by 25.1%and 37.2%,respectively.These data indicated that OsACL5 negatively control rice grain yield.6.We observed the seedling emergence rate of wild-type and transgenic lines from different soil depths.Only 66.5%wild-type seedlings had emerged from a soil depth of 5 cm after 9 days.The emergence rates of OsACL5-OE-1 and OsACL5-OE-2 lines were 41.7%and 52.7%,respectively.However,the emergence rates of osacl5-1 and osacl5-2 mutants were 80.3%and 83.0%,respectively.These results indicated that OsACL5 negatively regulates seedling emergence rate of direct seeding.We further found that the mesocotyl and coleoptile in osacl5 mutants were significantly longer than that in wild-type.We infer that OsACL5 control emergence rate may through regulating the elongation of mesocotyl and coleoptile.