Study On The Role Of NtGBSS2,4 And NtPTST4 Genes In Starch Synthesis Of Leaves In Nicotiana Tabacum

Tobacco(Nicotiana tabacum L.)is an important non-food economic crop that uses leaves as its harvest product.When tobacco leaves mature,the content of starch can reach40%.Starch synthesis in leaves involves the coordinated action of various enzymes.First,ADP-glucose pyrophosphorylase(AGP)catalyzes the production of adenosine diphosphate glucose(ADPG)from glucose 1-phosphate(Glc-1P)as the substrate for starch synthesis.Then,different starch synthase(SSs)catalyze the transfer of the glucose unit of ADP-glucose to the glucan chain to form α-1,4 bond.Starch synthase can be broadly divided into two categories: granular bound starch synthase(GBSS),which synthesizes amylose,and soluble SSs containing multiple subtypes that participate in the synthesis of amylopectin.Starch branching enzyme(SBE)passes the glucan short chain synthesized by soluble SSs through α-1,6 glycosidic bonds are attached to long chains,which together form amylopectin.Protein Targeting to Starch(PTST),which has been reported in recent years,is also involved in the synthesis of amylose.PTST can not only help locate GBSS in Arabidopsis onto starch particles,but also is crucial for starch synthesis in barley endosperm.At the same time,its homologous genes also play a decisive role in the initiation,quantity,and size of starch particles in Arabidopsis.The composition of starch,the ratio of amylose to amylopectin,and the size of starch particles can all affect the starch degradation rate.Starch content is one of the important factors that affect the curing characteristics of tobacco.Whether starch is fully degraded and the sugars after hydrolysis can affect the color,aroma,and taste of tobacco leaves.Except for the overexpression of NtAGPase and NtGBSSⅠ in tobacco,which can lead to starch accumulation,there are no other reports on starch synthesis and regulation in tobacco leaves.Whether GBSS and PTST related genes are involved in amylose synthesis in tobacco remain unknown.In this study,tobacco Honghua Dajinyuan was selected as the research object to screen the homologs of amylose synthesis genes Granule Bound Starch Synthesis(GBSS)and Protein Targeting to Starch(PTST).NtGBSS and NtPTST were knocked out by genetic engineering methods,and corresponding mutant plants were obtained.Their starch content,starch particles,and related carbohydrate substances were investigated.The following are the results of this study:1.Identification and expression characteristics analysis of NtGBSS and NtPTST homologs in N.tabacumIn order to identify amylose synthesis genes in tobacco,we first used the amino acid sequences of granular bound starch synthase and protein targeted starch reported in the literature as the query seed sequences,and compared them across the entire tobacco genome to screen candidate genes for granular bound starch synthase and protein targeted starch.Based on the results of sequence similarity and functional domain analysis,a total of 8 NtGBSS candidate genes(NtGBSS1 to NtGBSS8)and 7 NtPTST candidate genes(NtPTST1 to NtPTST7)were identified in tobacco Honghua Dajinyuan variety.For the NtGBSS gene family,the results of constructing evolutionary trees with other species indicate that the NtGBSS gene has high homology with the GBSS I of Arabidopsis,tomato,and potato.Through multiple sequence alignments,it was found that the NtGBSS gene contains multiple conserved amino acid sites with GBSS functional structures,including the ATP binding sites K(lysine),G(glycine),I(isoleucine),R(arginine),F(phenylalanine),A(alanine),E(glutamic acid)of glycosyltransferase family 5,as well as S(serine),V(valine)of glycosyltransferase family 1 with GTB topological structure,D(aspartic acid)sites,these active sites mainly function as glycosyltransferases.Gene structure analysis showed that the NtGBSS gene contains multiple exons,ranging from16 to 30,and genes located in the same branch have similar structures.Tissue expression profile analysis showed that except for NtGBSS3,which was not expressed in seeds,other genes were expressed in all tissues,with NtGBSS1,NtGBSS3,NtGBSS4,and NtGBSS5 being highly expressed in leaves,NtGBSS6,NtGBSS7,and NtGBSS8 being highly expressed in seeds and roots,and NtGBSS2 being the most highly expressed in seeds.As for NtPTST gene,evolutionary tree results showed that tobacco NtPTST gene has high homology with corresponding genes in plants such as Arabidopsis and tomato.NtPTST4,NtPTST5 has a close evolutionary relationship with At PTST2 in Arabidopsis.Multiple sequence alignment revealed that it contains multiple conserved amino acid sites:mainly N-terminal AMP activated protein kinase domain sites W(tryptophan),K(lysine),G(glycine),and N(asparagine).These active sites are glycogen binding sites that participate in the starch binding process.Tissue expression profile analysis showed that NtPTST1 to NtPTST7 were expressed in various tissues,with NtPTST1,NtPTST3,NtPTST6,NtPTST7 being highly expressed in seeds and roots,NtPTST2 being highly expressed in roots,NtPTST4 being the highest expression in leaves,and NtPTST5 being relatively high in stems.Based on the analysis of sequence and expression characteristics,NtGBSS2,NtGBSS4 and NtPTST4 may play an important role in the synthesis of amylose in tobacco leaves.2.Preparation of knockout plants for NtGBSS and NtPTST The complete CDS sequences of the three genes NtGBSS2,NtGBSS4,and NtPTST4 were obtained through PCR amplification.Then,a knockout vector was constructed using CRISPR/Cas9 gene editing technology.Mutants of the three genes were obtained through Agrobacterium mediated genetic transformation,and homozygous plants were obtained.The mutant plants obtained from NtGBSS2,NtGBSS4,and NtPTST4 were detected and the main forms of mutation were deletion and insertion.The amino acid sequence encoded by the mutated gene was analyzed,and the results showed that the amino acid sequence encoded by the mutant type gene with a multiple base deletion of 3 in NtGBSS2 and NtGBSS4 was partially deleted.The remaining mutant forms have resulted in early termination of the translation of NtGBSS2,NtGBSS4,and NtPTST4 genes,and the insertion of the terminator is located before the functional domain.3.Characterization of NtGBSS and NtPTST knock-out mutant plantsPhenotypic observation and related substance content detection were performed on the mutant plants of vigorous growth period.Compared with the wild type control,the growth and development of NtGBSS and NtPTST knockout tobacco were not affected.The detection of granular bound starch synthase(GBSS)activity showed that the enzyme activity of NtGBSS2-8 hybrid plants significantly decreased by 56.79%,while the enzyme activity of the other two hybrid plants and one homozygous plant of NtGBSS2 decreased but the difference was not significant;The enzyme activities of NtGBSS4-3 and 10 heterozygous plants of NtGBSS4 significantly decreased by 51.86% and 49.47%,respectively.The enzyme activities of NtGBSS4-9 homozygous plants significantly decreased by 55.12%,and there was no significant difference between NtGBSS4-6 and the wild type;However,the granular bound starch synthase activity of NtPTST4 mutant plants decreased but the difference was not significant.This may be because PTST gene is the target starch of GBSS gene,and its editing does not directly affect GBSS enzyme activity.Amylose content testing showed that the amylose content of the four mutant strains of NtGBSS2 significantly decreased by about 28.3%,50%,50%,and 30%,respectively,and NtGBSS2-6,NtGBSS2-8,and NtGBSS2-9 showed a very significant decrease;The amylose content of the four mutant strains of NtGBSS4 decreased significantly by 50%,30%,40%,and 38%,respectively;The amylose content of NtPTST4 mutants,whether homozygous or chimeric,decreased significantly,with a decrease rate of 26.6% to 58%.The amylose content of the three gene mutants decreased significantly or extremely significantly,and the decrease rate was related to the editing form and editing efficiency of different genes.The amylopectin content of mutant plants showed a trend of increasing or unchanged among different mutant plants.Except for the significant increase in amylopectin content of four mutant plants NtGBSS2-5,NtGBSS2-9,NtGBSS4,and NtPTST4-4,NtPTST4-6,NtPTST4-8,there was no significant difference among other mutant plants.Compared with the wild type,the total starch content of NtGBSS2-5,NtGBSS2-6,NtGBSS2-8,NtGBSS2-9 knockout plants significantly decreased by 24.43%,35.45%,47.86%,and 27.04%,respectively;The total starch content of NtPTST4 knockout plants decreased significantly by 33.97%,30.9%,19.04%,29.02%,12.21%,27.9%,and 26.17%,respectively;The total starch content of NtGBSS4 mutant was not significantly different from that of the wild type.We speculate that this may be related to the proportion of amylose and amylopectin changes,but the specific relationship needs further study in the future.Purified starch particles from leaves and observed with scanning electron microscopy,it was found that there were significant differences in the starch particle sizes of NtGBSS2 and NtPTST4 mutant plants.As a control,the starch particle sizes of the two wild type plants were both below 3μm,with the starch particles smaller than 1μm accounting for 7.25% and 10.6%,respectively.The largest proportion was between 1-2μm,accounting for 81.16% and 76.52%,respectively,while the remaining 11.59% and12.88% were between 2-3μm.In contrast,the starch particles in NtGBSS2 mutant strains significantly increased,with only 2.46%-4.46% of the starch particles in the four mutant strains being less than 1μm;Most of them are between 2-3μm,accounting for 41.98%-55.22% of different mutant strains;Some particles have a diameter of 3-4μm,accounting for 7%-18.6%;Among the NtGBSS2-5,NtGBSS2-6,NtGBSS2-8 mutants,more than1.23% of the starch particles were larger than 4μm in diameter.The starch particle size of NtPTST4 mutant was significantly increased.Except for NtPTST4-8,starch particles smaller than 1μm in other mutants ranged from 0.8% to 6.6%;Most starch particles are between 2-3μm in size,accounting for over 33.97%;More than 3.87% of all mutants have starch particle sizes greater than 3μm,while NtPTST4-3,NtPTST4-4,NtPTST4-5,and NtPTST4-7 mutants have starch particle numbers greater than 10%.The proportion of large granule starch in mutant plants was significantly greater than that of wild type,and the starch granules were more rounded.However,the size and structure of starch granules in NtGBSS4 mutant plants were not significantly different from those of wild type.The starch granules in NtGBSS4 were generally similar to those of wild type,with the largest proportion between 1-2μm,accounting for 65.91% to 74.38%;The proportion of starch particles smaller than 1μm in the 4 mutant strains was 6.82%-25.93%,higher than that in the wild type;Among NtGBSS4-6,NtGBSS4-9,and NtGBSS4-10,0.62%,0.75%,and2.27% of the starch particle sizes exceeded 3μm,respectively.It was found that the content of reducing sugar and glucose in the mutant plants of NtGBSS2,NtGBSS4,and NtPTST4 significantly increased,which is more conducive to the Maillard reaction with amino compounds.In summary,this study obtained mutant plants with NtGBSS2,NtGBSS4,and NtPTST4 genes through CRISPR/Cas9 gene editing technology.The experimental results showed that NtGBSS and NtPTST4 were involved in the synthesis of amylose in tobacco leaves.Mutating of some NtGBSS and NtPTST genes led to a significant change of size and structure of starch particles,and could affect the content of reducing sugar and glucose in the leaves.The research work has laid the foundation for a comprehensive understanding of the starch synthesis and regulatory mechanisms in solanaceous plants,and also provided a new perspective for improving tobacco leaf quality through genetic engineering.