Response Of Light And Fertilizer Regulation On The Growth Physiology And Main Secondary Metabolites Of The Hybrids Taxus Madia × Taxus Yunnanensis ‘Yunman’

Taxus is the only material required for the industrial production of the anticancer drug paclitaxel and 10-Deacetylbaccatin III（10-DAB）,the ingredient required for artificial semi synthesis.Taxus madia×Taxus yunnanensis‘Yunman’（YM）is a new medicinal plant variety of Taxus with high concentration of 10-DAB selected and bred by the research group through artificial hybridization.The F₁ hybrids seedling（<5 years old）has obvious heterosis,but only has 4～5 years of available period,resulting in low biomass of individual plant,low accumulation of major secondary metabolites at harvest time and insufficient yield per unit area,which hinders the utilization of heterosis and promotion of the variety.Boosting the growth of seedlings by artificial regulation is the most simple and convenient measure to promote the large-scale planting of new varieties,which is revealing the response mechanism of artificial regulation to the growth of seedlings and secondary metabolites.Therefore,exploring an effective regulation measure to increase the accumulation of major secondary metabolites per plant during the utilization cycle is a scientific and technical problem to be settled urgently in terms of the utilization of heterosis and promotion of the variety for YM.Based on the research of the biological characteristics of YM in the early stage,and taking 1-year-old container seedlings before leaving nursery as experimental materials,this paper carries out growth surveys,physiological and biochemical indicators detection,and dynamic tracking measurement of secondary metabolites with the method of steady state nutrient fertilization,so as to study the influence of nutrients on the growth,nutritional physiology,and main secondary metabolites and reveal the laws of fertilizer requirements for growth and the characteristics of nutrient accumulation and distribution.2-year-old seedlings after leaving nursery are used as experimental materials for the study on the growth,photosynthesis,stress resistance physiology,and accumulation response of major secondary metabolites of seedlings under the conditions of light and fertilization interaction.Based on the construction of the optimized model of the light and fertilization interaction mode with the fuzzy Borda method,as well as transcriptome sequencing and analysis,it explores the physiological and molecular response mechanism of the growth and light and fertilization compound control of the secondary metabolites of YM,and seeks effective measures for boosting growth by artificial regulation.Our studies provide theoretical and technical support for the utilization of heterosis and variety promotion.The main research results are as follows.（1）Steady state nutrient fertilization can significantly promote the biomass and accumulation of nutrient elements N,P,and K in 1-year-old seedlings of YM（P<0.05）.The biomass and nutrient accumulation reach highest with a nitrogen application rate of 1.6g/plant under steady state nutrient fertilization.When the nitrogen application rate is higher than 3.2 g/plant or lower than 2.4 g/plant,the growth and the synthesis of taxanes will be inhibited.Taxol and 10-DAB in branches and leaves present an upward trend from May to September,and a downward trend from September to November.The distribution amount of nutrients N,P,and K in various organs of 1-year-old seedlings of YM is in the order of leaves>roots>stems.The overall trend of nutrient element absorption in leaves is displayed as N>K>P,and there is a significant positive correlation between P and K,P and N in various organs,as well as N and K in leaves and roots（P<0.05）.（2）The combined regulation of light and fertilizer significantly improves the photosynthesis of 2-year-old seedlings of YM,promotes the synthesis of soluble proteins,soluble sugars,and proline,as well as the accumulation of taxanes（P<0.05）.Under conditions of 40～70%light and nitrogen application of 3.6 g/plant,the cumulative amount of paclitaxel and 10-DAB in branches and leaves of single plant significantly increases（P<0.05）.The rapid growth period of 2-year-old seedlings of YM is from March to June,and the optimal harvesting and utilization period is from September to December.The cumulative amount of paclitaxel and 10-DAB of single plant reach their maximum values in September（9.010 mg）and December（85.228 mg）,respectively.（3）From three aspects of physiology,growth,and secondary metabolism,12 key indicators such as net photosynthetic rate（P_n）,stem volume index（SVI）,and paclitaxel accumulation（APTX）are selected to establish a comprehensive evaluation index system.The improved CRITIC method,Grey Relational Analysis,TOPSIS method,and Membership Function Analysis method are used for the comprehensive evaluation and ranking of 16 light fertilizer regulation combinations.Theλ² testing method is used to verify the consistency of the four evaluation methods,and construct a optimized model of the light and fertilization combination of YM with the fuzzy Borda method.According to the result,the fuzzy Borda combination evaluation values above 90 points are in an order of S3F3（40%light+3.6 g/plant nitrogen application）>S2F3（70%light+3.6 g/plant nitrogen application）>S3F2（40%light+1.8 g/plant nitrogen application）.（4）Through the analysis on the leave transcriptome of 2-year-old seedlings of YM in the light and fertilization optimal group S3F3 and the control group（untreated）,a total of1,334 DEGs（differentially expressed genes）are identified,including 752 up-regulated genes and 582 down-regulated genes.All 569 transcription factors such as b HLH,NAC,MYB are identified in DEGs.21,944 transcripts have the highest annotation rate in the Nr database,saying 75.40%.The gene ontology（GO）enrichment of differential genes mainly lies in signal transduction,transmembrane transport protein activity,defense response,etc.Kyoto encyclopedia of genes and genomes（KEGG）enrichment mainly occurs in the aspects of biosynthesis of phenyl-propane,diterpene,and terpenoid frameworks.The light and fertilization compound control induces differential expression of transcription factor genes like b HLH and NAC,which affects the growth physiology and secondary metabolite synthesis of YM mainly through phenylpropanoid metabolism,starch and sucrose synthesis,and terpenoid biosynthesis pathways,in response to the effect of light and fertilization interaction.Among them,five CPX genes affecting paclitaxel synthesis are identified.In summary,there is a significant effect of steady-state nutrient fertilization on the improvement of biomass accumulation of 1-year-old container seedlings of YM.The light and fertilization combination S3F3（40%light+3.6 g/plant nitrogen application）can significantly promote the cumulative amount of paclitaxel and 10-DAB of single plant in 2-year-old seedlings of YM.The research result reveals the growth and metabolic response mechanism of YM seedlings under light and fertilization regulation.This finding provides theoretical and technical support for the utilization of the heterosis of the medicinal plant YM,and the promotion of varieties.