Study On The Mechanisms Of Gibberellin Controlling Plant Height Architecture In Agapanthus Praecox Ssp.Orientalis

Plant height is one of the most important phenotype affects plant production and quality in the field of crop and horticulture,and dwarfism is a favourable traits in ornamental plants application.Agapanthus praecox ssp.orientalis is a perennial bulbaceous plant,mainly distributed in the southern African continent,and is popular as a landscape plant because of its high ornamental value.The length of leaves represents its plant height in seedings,whereas the length of scape（stem inflorescence）determines its height in the adult A.praecox plants.In the previous study,we found that gibberellin（GA）is the key regulator in plant height determination,but little information for the mechanisms of plant height architecture is available.In this study,the morphological and physiological effects of exogenous GA₄ and paclobutrazol（PAC）on 1-year seedlings and 5-year adult plants were investigated in A.praecox,and results showed that the scape height reduced significantly with PAC treatment.Then we elucidated the mechanisms of scape dwarfing by comparative transcriptomics,and the results suggested that ApGA20ox1 was crucial in scape height control.The expression of ApGA20ox1 was inhibited by RNA interference（RNAi）,and the transgenic RNAi seedlings presented severe dwarf phenotype.The mechanisms of seeding dwarfism regulated by GA signal were investigated by combined morphological,physiological and proteomics data.The results of this study were outlined below.（1）In order to investigate the regulatory effects and the physiological basis of GA signals,the 1-year seedlings and 5-year adult plants were sprayed with 400 mg·L^-1GA₄ and PAC respectively.Results showed exogenous spray of GA₄ has no significant effects on A.praecox height determination,and GA₄ has no significant influences on chlorophyll content and the net photosynthetic rate（Pn）.The contents of starch reduced 8.70%and 42.28%in seedlings and adult plants,and the contents of soluble sugar decreased 11.99%in scape with GA₄ treatment.Besides,the fatty acid contents in seedling leaves and scape decreased 16.26%and 21.31%with GA₄treatment respectively.On the contrary,the length of leaves was reduced 52.40%in seedlings,and decreased 36.22%and 54.56%in leaves and scape respectively in adult plants with PAC treatment.Microscopic observation showed that PAC reduced plant height mainly by inhibiting cell longitudinal size,and the cell length reduced 36.10%in seedling leaves,and decreased 31.27%and 50.91%in leaves and scape respectively in adult plants compared with control.Compared to cell length,PAC has negligible influence on cell width.Physiological investigation showed that the contents of chlorophyll were increased 29.92%and 18.45%,and Pn was enhanced 14.99%and21.16%in seedings and adult plants respectively.The contents of starch increased18.98%and 30.68%,and the amounts of soluble sugar were also increased 68.08%and 17.81%in seedlings and adult plants respectively.The results above indicated PAC was more effective than GA in A.praecox plant height determination,espcially in scape height control,and the results also proved the accumulation of assimilation products possibly involved in the regulation of plant dwarfism.（2）Comparative transcriptomics analysis was investigated in control and PAC-treated dwarfing scape,and totally 2838 dwarf related differentially expressed genes（DEGs）were identified.These DEGs were significantly enriched in hormonal signaling,carbohydrate metabolism,cell circle and division,cell wall synthesis and degradation related pathways.Then the concentrations of hormones including GA₁,GA₃,GA₄ and indole acetic acid（IAA）,starch and sucrose,the components of cell wall,and gene relative expression levels were investigated in top,middle and bottom of scape respectively.The results indicated that GA₄ was the main component of GAs in scape.After PAC treatment,the contents of GA₁ and GA₃ decreased 3.41-65.43%and 5.06-61.43%respectively and the concentration of GA₄ decreased dramatically by59.5-92.0%in scape.Furthermore,the top/bottom ratio of IAA contents decreased from 14.57 to 1.77 after PAC treatment.GAs determined cell longitudinal size（correlation index was 0.822）,whereas IAA regulated the expression of Cyclin-SDS（correlation index was 0.980）that reduced the cell number by 10.45%.The output of cell wall components such as cellulose and hemicelluloses increased significantly,whereas the transcriptional levels of cell wall loosening related genes such as xyloglucan endotransglycosylase2（XET2）and for touch（TCH4）were decreased99.99%in dwarf scape.The expression of XET2（correlation index was 0.814）and TCH4（correlation index was 0.897）were regulated by GA₄,and the biosynthesis of GA₄ was regulated by ApGA20ox1（correlation index was-0.870）,suggested that ApGA20ox1 is a key regulator in A.praecox height control.（3）The full length cDNA of ApGA20ox1 was obtained by RACE technique.ApGA20ox1 was consisted by three exons and two introns,and full length cDNA was1386 bp.The 1086 bp of open reading frame（ORF）encoding a putative polypeptide of 361 amino acids.Phylogenetic analysis revealed that ApGA20ox1 was highly homologous with GA20ox in monocotyledons.The real-time quantitative PCR test indicated that ApGA20ox1 was accumulated in root tips,immature seed,stem and scape,suggested ApGA20ox1 is critical to the development process in A.praecox.Sub-localization experiment showed ApGA20ox1 performed its functions as a cytoplasmic protein.Over-expression of ApGA20ox1 in Arabidopsis thaliana showed that ApGA20ox1 increased 21.77%and 20.74%in rosette leaves and the plant height,indicated that ApGA20ox1 has the biological function in plant height determination.（4）By inhibiting the expression of ApGA20ox1,RNAi transgenic plantlets were induced successfully in A.praecox.The expression of ApGA20ox1 was inhibited by39.48%in 2-year transgenic RNAi plants leaves,and leaves length reduced considerably by 55.59%,indicated that ApGA20ox1 regulated plant height in A.praecox.Microscopic observation showed that RNAi plantlets reduced plant height mainly by inhibiting cell longitudinal size,and the cell length reduced 44.03%compared with control.Exogenous spray of GA₄ rescued the dwarf phenotype,and the amount of leaves length growth increased 208%compared with control,suggested that the dwarf trait was attributed to GAs deficiency.The contents of GA₁,GA₃ and GA₄ decreased 37.49%,53.82%and 49.68%in RNAi dwarf plants respectively.The contents of brassinosteroids（BRs）and jasmonate（JA）were also decreased 48.28%and 39.88%,whereas the content of IAA was increased 207.04%in RNAi plantlets.Furthermore,the contents of starch and soluble sugars increased 48.69%and 120.38%in dwarf plants.The results suggested that GA signals regulated the synthesis and metabolism of other plant hormones and sugars in A.praecox seedlings.（5）Differentially expressed proteins（DEPs）were screened in RNAi dwarf plants by comparative proteomics,and the 55 DEGs were significantly enriched in hormonal synthesis and metabolism,carbohydrate metabolism,secondary metabolism,cell wall synthesis and degradation,and stress response related biological processes.The results of comparative proteomics were mainly in accordance with the comparative transcriptomics analysis in dwarf scape.Pathway analysis indicated that GA was the main regulator in plant height control,and IAA,BR and JA signals were regulated by GA signal,and synergistic regulated these biological processes.The correlation analysis was conducted by employing the data of comparative transcriptomics and proteomics,and the results showed that DEGs and DEPs were significantly enriched in glycometabolism,cell wall synthesis and degradation.DEPs including fructose-1,6-bisphosphatase,fructose 6-phosphate 1-phosphotransferase,alpha-glucosidase and alpha-galactosidase were up-regulated,enhanced the accumulation of sugars,which provides abundant precursor in cell wall structure synthesis.Furthermore,the up-regulation of cellulose synthase,UDP-glucuronic acid decarboxylase and xylan 1,4-beta-xylosidase strengthened the structure of cell wall,whereas the down-regulated of XET2 and TCH4 reduced the cell wall modification and degradation.Consequently,the plasticity of cell wall was inhibited,and the size of cell wall was reduced,and the plant represents a dwarf phenotype in A.praecox.Base on these results,we predicted and constructed a regulatory network of GA signal,which elucidated the mechanisms of GA controlling plant height architecture in A.praecox preliminarily.