Analysis Of The Association Between Albino Mutant And Photosynthetic Pigment Anabolism And Drought Tolerance In Daylilies

Daylily(Hemerocallis spp.)is a perennial herbaceous flower,which has been cultivated in China for thousands of years and has high breeding value.The daylily natural albino mutant was found during screening the self-crossing offspring of the daylily cultivar’Black-eyed stella’,.This would be help for the future development of daylily and for increasing understanding of photosynthesis mechanism.In order to explore the physiological characteristics of the mutant and the role of the mutant gene,a variety of research methods was used in this study,and the main research results are as follows.(1)Leaves were light yellow and the height of the albino mutant plants was lower than that of the wild type.The mutant plants survive for less than 15 days when cultured in soil,but survive longer when cultured in medium containing sucrose in sterile conditions.Investigation by optical microscope and transmission electron microscope showed that the number of chloroplasts in the mutant was significantly less than that of the wild type,Compared with that of the wild type,the chloroplast structure of the mutant had some empty bubbles,and the number of cystoids and the degree of accumulation in the grana of the mutant was significantly reducedin mutant plants.(2)Thedaylily ’Black-eyed stella ’is the F1 generation of the cross between ’Stella de oro’(diploid)and ’Little celena’(diploid)’.The segregation ratio of selfed progeny of ’Black-eyed stella ’ was investigated.I t was found that the ratio of wild-type and albino mutant plants in F2 was 3:1.Self-crossing experiments were also performedusing F2-generation plants.It was found that some of the F2-generation plants don’t have albino self-progeny,these plants were marked as Alb+/+.And other F2-generation plants still had about 30%albino self-progeny,these plants were marked as Alb+/-.The a forementioned results suggest that the albino mutation was controlled by a single recessive gene.(3)The total chlorophyll content was reduced by 99.45%,chlorophyll a content by 99.43%,chlorophyll b content by 99.58%,and carotenoid content by 97.89%in the mutant compared with the wild type.The content of chlorophyll precursors was significantly reduced except for bilirubininogen(PBG).However,the genes encoding the enzymes catalyzing biosynthesis of bilirubinogen(PBG)from 5-aminolevulinic acid(ALA)were not down-regulated,which suggested that this mutated gene was upstream of biosynthesis pathway of chlorophyll and carotenoid.(4)The analysis of transcriptome between wild-type and albino plants showed that there were 7952 differential genes,including 4069 up-regulated genes and 3883 down-regulated genes.Analysis of the metabolic pathways of differential genes(DEGs)and Kyoto Encyclopedia of Genes and Genomes(KEGG)showed that DEGs related to chlorophyll biosynthesis were mostly down-regulated,and there were also down-regulated DEGs upstream of the 2-C-methyl-D-erythritol-4-phosphate metabolic pathway(MEP)and mevalonate metabolic(MVA).The transcriptome sequencing results were verified by real-time PCR.These results implied that this mutated gene lead to a decrease in chlorophyll and carotenoid content,and seriously affect the normal growth and development of plants.Based on plant phenotype,chlorophyll and its precursor content,transcriptome assay analysis and the previous research papers,it is speculated that the gene encoded 1-deoxyxylulose-5-phosphate synthetase(DXS)may be the mutated gene,which is responsible for the mutant phenotype.(5)The determination of photosynthetic pigments and photosynthetic parameters using Alb+/+and Alb+/-plants showed that the chlorophyll(Chl),carotenoids(Car),intercellular carbon dioxide concentration(Ci),net photosynthetic rate(Pn),stomatal light conductance(Gs),transpiration rate(Tr)and light saturation point of Alb+/+ plants were significantly higher than those of Alb+/-plants,indicating that the photosynthesis rate of Alb+/+was significantly stronger than that of heterozygous Alb+/-plants.It indicates that the mutation of this gene causes obstacles to photosynthetic pigment synthesis and thus reduces the photosynthetic rate.(6)The drought treatment experiments followed by measurement of the leaf water content,relative soil water content,and photosynthetic parameters during drought were performed using Alb-/-,Alb+/+ and Alb+/-plants.One-week-old Alb-/-seedlings collapsed and their leaves wilted and bent more quickly compared to wild type plants.For 2 year old plants,photosynthetic parameters such as cell Intercellular CO2 concentration(Ci),net photosynthetic rate(Pn),water use efficient(WUE),and transpiration rate(Tr))were significantly higher in Alb+/-plants than in that in Alb+/-plants.Further more,the relative leaf water content was higher in Alb+/+plants than in Alb+/-plants,presumably the homozygous plants were able to produce more photosynthetic pigments compared to heterozygous plants.And this gene mutation resulted in drought sensitivity.