Sorus Formation Of Saccharina Japonica: Optimization Of Conditions, Biological Process And Regulatory Mechanism

The sorus（fertile sporophyte）of kelp Saccharina japonica is an important reproductive organ for sexual reproduction and population continuity.The production of sorus in appropriate season is an important guarantee for the development of kelp breeding industry.New kelp varieties/strains bred by hybridizing tend to mature late,resulting in delayed sorus formation,which cannot meet the normal needs of kelp seedling production.In this paper,the culture conditions for promoting the sorus formation of S.japonica were screened and optimized,and the apparent characteristics,tissue structure,physiological biochemistry,hormone levels and gene transcriptional expression were systematically studied during the development of sorus.The aims of this study were to reveal the conditions and requirements,biological process and regulation mechanism of S.japonica sorus formation.The primary research results are as follows:Temperature was the foremost environmental factor affecting sorus development,and sporangia form the fastest when cultured at 17°C.These factors（for example:light intensity,nitrogen and phosphorus concentration,abscisic acid,sporophyte transection treatment,etc.）could all affect the formation of sorus.Whether the concave surface of the kelp sporophyte receives light is independent of the formation of the sorus,indoor sporophytes produce sorus more quickly on the backlit surface.The addition of Fe³⁺delays the formation of sorus and may promote decay of kelp sporophytes.The rapid formation of sorus can be promoted by controlling the temperature of 17°C,nutrient salt concentration of N 5 mg/L+P 0.5 mg/L,abscisic acid concentration of 10^-6-10^-5 mol/L,light cycle 8:16（L:D）,and light intensity of 60-100μmol·m^-2·s^-1.From the thickening period to the stage of sorus formation,the apparent characteristics mainly undergoes the surface of the sporophyte from smooth to frosted and produces a frosted bump,followed by a thickening of the bulge,deposit at apex of the paraphysis cells falls off,and the surface of the sporophyte becomes smoother.Meanwhile,the epidermal tissue differentiates to form paraphyses which continues to prolong in the stage of sorus formation,and the basal cells differentiate to form spore mother cell and develop zoospores.The process was accompanied by an increase in soluble proteins,the accumulation of chlorophyll and nitrogen content,and the ROS such as hydrogen peroxide and superoxide anions.The overall enzymes activities of SOD,APX,POD,and CAT showed a decreasing trend,however,the enzymes activities showed different degrees of increase in different stages of sorus formation,and it was no significant change in malondialdehyde content.The sorus development of S.japonica was regulated by a variety of hormones.During the sorus formation,content of similar hormones varies greatly,and the response of different hormones was not completely consistent.Indole-3-acetic acid（IAA）and gibberellin 7（GA7）were higher in S.japonica.With the sorus forming,the content of salicylate（SA）and gibberellin 1（GA1）increased;Indole-3-acetic acid and gibberellin3 levels decrease at the beginning of sporangia and then begin to rise.In addition to the increase in trans-Zeatin（t Z）,cytokinin content such as trans-Zeatin-riboside（t ZR）,Dihydrozeatin（DHZ）,N6-Isopentenyladenine（i P）and Isopentenyladenine riboside（i PR）were significantly reduced.Transcriptional level analysis of different stages showed that there were 906 upregulated genes and 626 downregulated genes for SA-vs-SB,and 2219 upregulated genes and 467 downregulated genes for SB-vs-SC.It was the main stage of gene regulatory changes,accompanied by the elongation of kelp epidermal cells,the formation of paraphyses and spore mother cells,and the process of transformation into sorus.SC-vs-SD,where there are only 288 upregulated genes and only 27downregulated genes,was the continuous development of the sorus and promotes epidermal shedding;SD-vs-SE,with 237 genes upregulated and 152 genes downregulated,was the stage from no zoospores to the release of zoospores after epidermal detachment.The GO function annotation found that genes of sustained high-throughput expression were mainly enriched in membrane synthesis,cytoskeleton,chromosomal changes,and catalytic activity related metabolism,whereas genes of continuous downregulated were mainly enriched in oxidation-reduction,single-organism catabolic,carbohydrate catabolic process,organonitrogen compound metabolic process.moreover,the KEGG pathway enrichment analysis showed that the metabolic pathways such as MAPK signaling pathway-plant,purine metabolism,plant-pathogen interaction,and plant hormone signal transduction were significantly upregulated during sorus development.This indicated that plant hormone signal transduction and MAPK signaling pathway-plant genes were involved in the sorus development.In addition,22 genes related to carbon metabolism pathways were significantly downregulated during the development of sporangia,which may be related to the reduction of the accumulation of carbon sources during sorus formation,and the downregulation of redox metabolism-related gene expression may be attribute to the decrease in redox-related metabolic activities.