| Sugarcane(Saccharum spp.hybrid)is a noteworthy crop in the world's economy,renowned universally for sugar production and higher capacity of sucrose accumulation in stalks.It is well cultivated commercially in at least 106 countries of tropical and subtropical regions of the world,which produces around 70% of global sugar.To meet the increasing needs of sugar,it becomes significant to develop sugarcane varieties with higher sucrose yield.In the present study,a high sucrose content sugarcane mutant clone,GXB9,has been studied in comparison to the low sucrose mother variety B9 on morphological,physiological,agronomic,transcriptomic and proteomic level.The sugarcane variety B9 was introduced from Brazil in October 1999,which showed wide adaptability,high yield but relatively low sugar content.On 15 th October 2013,a high sugar content clone was found in a population of B9 variety and it was given a name as GXB9.Since then,several years of field experiments have been conducted to compare the performance of GXB9 with its mother variety B9.The results proved that both clones have close morphological characteristics,but showed significant difference in sucrose content consistently,which suggests that mutation has occurred in GXB9.In order to scrutinize the alteration cause of mutation in GXB9,a schematic study was designed to locate the variation in morphological,physiological,and agronomic traits of GXB9 in comparison to B9,in the field under normal environmental condition.Further,Illumina Hiseq2000 transcriptomic and TMT quantitative proteomic approaches were adopted for molecular characterization of GXB9 in comparison to B9 at sugar accumulating and processing maturation stage to explore the genes and proteins associated with sucrose metabolism and accumulation.SSR polymorphism was also examined.The results of morphological and physiological investigations showed numerous variations in GXB9 compared to B9 in all the studied parameters.GXB9 has less germination and tillering rate than B9.Several other interesting and significant variations were noticed like plant height,leaf length,leaf width,leaf area,number of internodes,internode length,internode diameter,were higher in B9 than GXB9.Similarly,variations were noticed in leaf sheath color,leaf sheath spines,wax content,dewlap color and shape,collar hairs,auricle shape and color,internode color,internode pith,growth cracks,bud shape and size,bud groove and position between GXB9 and B9.Comparative quantification of phytohormones also showed differences between GXB9 and B9.The concentration of gibberellic acid was significantly higher at grand growth stage in GXB9 than B9.The cytokinin endogenous hormone quantity was surprisingly significantly higher in B9 than GXB9 at tillering stage.The relative chlorophyll content SPAD and chlorophyll a,b concentrations between GXB9 and B9 showed certain variation but non-significant.The photosynthesis component analysis indicated that the GXB9 has significantly higher net photosynthetic rate(Pn),stomatal conductance(gs),transpiration rate(Tr)than B9.The qRT-PCR analysis of enzyme genes like cellulose synthase gene Ce S,cell wall invertase gene CWIN,sucrose synthase gene Su Sy,and sucrose phosphate synthase gene SPS also showed variations between GXB9 and B9.The relative expression analysis showed that the enzyme genes related to sucrose metabolism and accumulation,such as Ce S,CWIN,Su Sy,and SPS were upregulated in GXB9,and they had higher expression level than those in B9.The Su Sy showed higher expression level in the immature internodes than the maturing internodes in GXB9.The expression level of SPS was higher in the maturing internodes than immature internodes in GXB9.The immature internodes had a much higher expression level of CWIN than the maturing internodes in GXB9.Ce S had a higher expression level in the immature internodes than in maturing internodes in GXB9.The cane quality trait analysis showed that GXB9 had higher juice rate,juice gravity purity,Brix,juice sucrose content and cane sucrose content than B9.The yield and component investigation results showed that GXB9 had lower single stalk weight but higher number of millable stalks per hectare than B9,and GXB9 had lower theoretical cane yield than B9.The results obtained from the SSR marker analysis in the present study also showed genetic differences among the sugarcane genotype GXB9,B9 and GT32.Transcriptomic analysis was conducted for GXB9 in comparison with B9 at sucrose accumulating and mature stages.At sucrose accumulating stage transcriptomic analysis produced a total of 1,12,170 unigenes,and 1,06,026(94.52%)were annotated by the available public databases NR,GO,COG,KEGG,Swiss-Prot,and Pfam,while at mature stage a total of100,262 unigenes were obtained,and 69,637(69.46%)unigenes were annotated by the same databases.Functional assignment analysis of unigenes exhibited that they were active in a diversity of metabolic pathways.The differentially expressed unigenes(DEGs)for sucrose metabolism and accumulation were relatively characterized in the immature internodes(int.5,6)and maturing internodes(int.13,14)of GXB9 in comparison with B9.Sucrose phosphate synthase(SPS,EC 2.4.1.14),sucrose synthase(Su Sy,EC 2.4.1.13),and invertase(INV,EC 3.2.1.26)are vital enzymes,functional in the biosynthesis and splitting of sucrose in sugarcane.For sucrose phosphate synthase(SPS1,SPS2,SPS4,SPS5),the expressions of the DEGs were significantly upregulated in the maturing internodes of GXB9 and downregulated in B9.For Su Sy and invertase(CWIN,A/NINV),the expressions of the DEGs were upregulated in the immature internodes of GXB9 majorly,while the expressions of the DEGs involved in trehalose synthesis were downregulated in the immature and maturing internodes of GXB9 and upregulated in B9.Upregulation of the expressions of DEGs encoding SPS in the maturing internodes of GXB9 while downregulation in B9 was related to the higher sucrose accumulation in GXB9,which may be because of mutation.Further the downregulation of trehalose related DEGs in GXB9,and upregulation in B9,may be due to mutation.It may also be related to the higher sucrose accumulation in GXB9.Proteomic exploration of GXB9 in comparison to B9 through TMT quantitative approach produced a sum of 27,922 proteins.Among them a total of 7,484 proteins received functional assignment on blasting in COG,GO,Nr,Pfam,and KEGG databases.Collectively 3,102 differentially expressed proteins(DEPs)were recognized in the immature internodes(int.5,6)and maturing internodes(int.13,14)of GXB9 in comparison to B9.SPS,Su Sy and invertases are the significant proteins associated with sucrose metabolism and accumulation in sugarcane.The SPS proteins SPS2,SPS5 were upregulated,and Su Sy,CWIN,NIN1,CINV2 proteins were downregulated in the maturing internodes of GXB9 as compared to B9.Analysis of the data obtained from morphological,physiological,agronomical and molecular study as well as transcriptomic and proteomic characterization proved significant variation in GXB9 in comparison to B9.It confirms that beneficial spontaneous mutation has occurred in GXB9 clone,which resulted in the production and accumulation of higher sucrose content with several other modifications in morphological,physiological and agronomic traits.So,it is confidently concluded that GXB9 is a mutant,and higher expression of SPS has predominantly enhanced its capacity to accumulate higher sucrose concentration in stalks compared to B9. |
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