Relationships Between The Expression Patterns Of Genes Associated With Carotenoid Metabolism And The Formation Of Millet Color

Foxtail millet(Setaria italica(L.)Beauv.)is originated from China,and is an important minor crop.Hulless grains of foxtail millet is called millet,and millet color is an important indicator to evaluate foxtail millet quality,but the formation mechanism of millet color remains unclear.In this study,yellow(Jingu 21,Changnong 35)and white(Zhishenggu,Niumaobai)foxtail millet cultivars were selected as research materials to study the relationships between the content of total carotenoids and the formation of millet color,as well as lutein and zeaxanthin.The expression patterns of 14 genes related to the carotenoid pathway at different filling stages of grains and the sequence alignment of SiPSY1 gene were analysed to reveal the mechanism of the formation of millet color in both physiological level and the molecular level.The main results were:1.Through the determination of millet color with main indicators from CIE L*a*b*color system,the results showed that the yellower the millet,the higher value of b*,which indicated b*value was a good index to identify the differences of millet color.b*value had a significant negative correlation with L*(r=-0.702;p<0.05),and a significant positive correlation with a*(r=0.783;p<0.01),but there was no obvious correlation between b*and CCI(citrus color index).2.Spectrophotometer and HPLC were used to dertermine the total carotenoids,lutein and zeaxanthin content at different filling stages of foxtail millet grains,the results showed that main carotenoid components were lutein and zeaxanthin(both accounted for about 90%of total carotenoids)in millet,especially,lutein content was dominant.And the content of total carotenoids were significant higher in yellow millet than in white millet.b*value also showed a significant positive correlation with the content of total carotenoids,lutein and zeaxanthin,respectively.3.The expression patterns of 14 genes related to carotenoid pathway during grains maturation were analysed by quantitative real-time PCR.The differential expression of SiPSYl arised at the S4 stage of foxtail millet cultivars,especially in Jingu 21 and Niumaobai,which showed a significant positive correlation with most genes related to carotenoid biosynthesis.However,SiPSY2 and SiPSY3 had no obvious relationships.So SiPSY1 might play a major role in yellow pigment accumulation of millet,and its differential expression became one of the main reasons for causing the differential accumulation of yellow pigment.SiCCD genes played a key role in the degradation of carotenoids,they presented a relatively stable expression in the early stages and a gradually rising in the later stages.The expression quantity of SiCCD1a had a significant negative correlation with the content of total carotenoids and lutein.The expression of SiCCDla was higher in white millet than that in yellow millet during all stages of grain development.Thus,the differential expression of SiCCDl was another reason for the differential of carotenoid accumulation and affected the formation of millet color.The expression of SiHYD increased gradually as grains filling went on,which resulted in a higher accumulation of zeaxanthin in the later filling stages of millet.In addition,SiHYD had a negative correlation with genes(SiLCYE.SiCYP97C)from β,ε-branch pathway.It was predicted that SiHYD might play an important role in β,β-branch pathway.4.Through the DNA cloning and sequence alignment analysis of SiPSYl from Zhishenggu and Niumaobai,the results showed that SiSY1-encoded protein was a stable hydrophilic protein,which was located in the chloroplast and contains a transmembrane domain.SiPSY1 DNA sequences in four foxtail millet cultivars were different.Compared with yellow foxtail millet cultivar,most insertion and deletion of large fragments were occurred in introns of white foxtail millet cultivar.In addition,the single base mutation(T to C)at the 2212 bp of the fifth exon caused an amino acid variation(phenylalanine to leucine)in SiPSYl of Niumaobai,but this mutation did not change the physicochemical properties,secondary and tertiary structures of protein,which suggesting that the mutation did not cause the change of protein function.It might be necessary to further analyze the expression differences from SiPSY-related regulatory genes or promoter sequences.In summary,on the physiological level,the accumulation of lutein and zeaxanthin was the main reason for the formation of millet color.On the molecular level,the carotenoids degradation caused by SiCCDl was the main reason for the formation of white millet.Especially the differential expression of SiCCDl and SiPSY1 in later stages of grain filling resuled in a declining carotenoid synthesis rate and an increasing degradation rate,leading to the different accumulation of carotenoids between yellow and white millet and affecting the formation of millet color.