Related Gene Identification Of Slow Growth And Development Of Switchgrass At Seedling Stage

In recent years,the extensive use of fossil fuels has made global warming increasingly become a common global concern.Finding affordable clean energy is the fundamental solution to the problem.Switchgrass(Panicum virgatum L.),a perennial C4 grass originating in North America,is considered one of the ideal bioenergy crops due to its wide adaptation range,poor tolerance and high biomass in above-ground parts.Switchgrass,a perennial C4 grass originating in North America,is considered one of the ideal bioenergy crops due to its wide adaptation range,poor tolerance and high biomass in above-ground parts.There are many propagation methods of switchgrass,such as cutting,tissue culture and seed sowing.Due to the limitation of technology and cost,convenient and cheap seed sowing is still the main mode of reproduction in production.However,for large-scale popularization and application,seed sowing also has some disadvantages.Among them,the most unfavorable thing is that the seedlings grow slowly and weakly in the year when the seeds are sown,which makes it difficult to resist the encroachment of weeds,drought and other adverse external environment at the seedling stage,and the success rate of switchgrass planting in the year when the seeds are sown is very low.As a result,the success rate of establishment and planting in the year of sowing is very low,which seriously affects the large-scale application and promotion of switchgrass in production.Although the reasons for the slow growth of switchgrass at seedling stage have been studied more than 20 years ago and have been successfully explored in seed size and dormancy,sowing depth,soil type,soil moisture,and competition with weeds,the mechanisms involved in the growth and development of switchgrass seedlings themselves have been less well studied,nor have they been studied in other perennial grasses.Our laboratory has previously studied the causes of slow growth and development of switchgrass seedlings at physiological,biochemical,and transcriptome levels.The results of physiological and biochemical data showed that the slow growth of switchgrass at seedling stage may be related to root development,antioxidant capacity and gibberellin.The transcriptomic data related to seedling growth indicate that switchgrass seedling developmental divergence genes include not only functional genes involved in several metabolic and physiological processes such as thiamine biosynthesis,gibberellin biosynthesis,energy metabolism and circadian rhythm,but also regulatory genes of transcription factor families such as HSF and GRAS.Based on the results of previous studies,this study studied and analyzed the functions and regulatory networks of genes and families related to the growth and development of switchgrass at the seedling stage,and used cytological methods to explore the reasons for the slow growth and development of switchgrass at the microscopic structure level.The results are as follows:1.Among the differentially expressed genes associated with slow growth and development of switchgrass seedlings,thiazole synthetase gene THI1,which is related to thiamine biosynthesis,was highly expressed in fast-growing switchgrass seedlings,suggesting that thiamine might be related to growth of switchgrass at seedling stage.We verified the effect of exogenous thiamine on the growth and development of switchgrass seedlings by soaking seeds with aqueous thiamine solution and spraying seedlings.The results show that exogenous thiamine may not promote switchgrass seedling growth under normal growth conditions.However,overexpression of the switchgrass thiazole synthase gene PvTHI1 in Arabidopsis significantly increased drought resistance in Arabidopsis,suggesting that thiamine may play an important role in seedling response to an adverse environment.2.Among the differentially expressed genes for transcription factors associated with switchgrass seedling stage development,4% belong to members of the GRAS family.In this study,98 GRAS family members of switchgrass were identified from the whole genome of switchgrass by GRAS domain,and were named PvGRAS1-PvGRAS98 according to their positions on chromosomes.The switchgrass GRAS family members were divided into 11 subfamilies based on their phylogenetic relationships.Meanwhile,the chromosomal localization,gene structure,cis-acting elements of promoter regions,and tissue expression of switchgrass GRAS family members were analyzed using bioinformatics methods.The results of cis-acting elements and tissue expression analysis indicated that GRAS family members,especially At PAT1 subfamily,may play a role in the response to stress at seedling stage in switchgrass.Then,the At PAT1 subfamily member gene PvGRAS9 associated with the seedling growth of switchgrass was cloned,and it was overexpressed in Arabidopsis.Our results showed that overexpression of the At PAT1 subfamily member PvGRAS9 in Arabidopsis significantly increased the proline content and SOD activity of Arabidopsis seedlings under salt and drought stress and significantly enhanced the ability of Arabidopsis to adapt to adverse environments.3.Among the differentially expressed genes in switchgrass seedling development,a member of heat shock transcription factors(HSF)family heat shock transcription factors was clearly differentially expressed in seedlings with different growth and development rates.The HSF family are key transcription factors in plants responding to abiotic stresses.In this study,we identified 48 HSF family members by both keyword and conserved structure search,which were named PvGRAS01-PvGRAS48 according to their chromosomal location.Based on the phylogenetic relationships,the switchgrass HSF family members are divided into three major groups(A,B and C).Of these,60.4% of the family members belong to class A.Then,bioinformatics methods were used to comprehensively analyze the basic characteristics,potential functions and evolutionary relationships of switchgrass PvHsfs family members from the aspects of protein physicochemical features,conserved domain and gene structure,subcellular localization,chromosome localization,cis-acting elements,and phylogenetic relationships.The bioinformatic analysis showed that reason for the expansion of the HSF family members are multiple chromosome doubling events and segmental duplication events during the evolution of switchgrass.Moreover,members of switchgrass HSF family not only participate in stress response such as low temperature and drought,but also participate in the regulation of growth and development processes such as light response.At different periods after heat stress treatment,different HSF family members of switchgrass showed different expression patterns,suggesting that they may play different roles in the heat response pathways.4.There may be three cytological reasons for the slow growth and development of switchgrass at seedling stage:(1)Abnormal development of Casparin strip in some seedling roots may affect the normal absorption and transport of water and minerals and the resistance of seedling roots to adverse soil environment;(2)The bulliform cell of some seedlings were significantly smaller,which may reduce the adaptability of seedlings to abiotic stress such as drought;(3)The number of mitochondria in vascular bundle sheath cells of some seedlings was small,which may have insufficient carbon fixation capacity,thus reducing their photosynthetic efficiency and affecting the growth and development of seedlings.In conclusion,this study explored the intrinsic mechanisms affecting the development of switchgrass seedling stage from the molecular biology and cytological levels,which provide a theoretical basis for studying the causes of slow development of switchgrass seedling stage,and provided a little genetic resource for the improvement of adverse traits of switchgrass by bioengineering.