| From“Cereal”to“Food”and then to the“Greater Food”,in order to meet the trend of changes in the food structure of the masses,diversified,nutritious and,healthy has become the basic requirements of food consumption,which has brought new opportunities for nutritious coarse cereals.Proso millet（Panicum miliaceum L.）is the oldest crop originating from the Yellow River Basin in the north of China,and as the first of the five grains,it has advantageous and characteristic crop in the arid and semi-arid areas in the new period due to its unique characteristics of drought-resistant,barrenness-resistant and high water-use efficiency.Proso millet grains are rich in starch,protein,fat,trace elements,minerals and polyphenols,and are gluten-free,making them ideal for people with gluten intolerance.However,the low yield,poor palatability,late start of research and few research results have seriously hindered the industrialization of proso millet.Nitrogen fertilizer is an important agricultural cultivation measure,which plays a vital role in increasing crop yield and improving quality.Systematic research on the effect of nitrogen fertilizer on the yield and quality of proso millet grains and exploring its regulatory mechanism on quality is of great significance to the high-quality and high-efficiency cultivation and industrialization of proso millet.In this study,two types of varieties with differences in amylose content,the waxy proso millet Zhengninghongnian（W139,with 1.99%amylose content）and the non-waxy proso millet Nei Mi 5（N297,with 21.86%amylose content）,were selected from 301 core germplasm resources of proso millet,and four nitrogen fertilizer levels of N 0 kg·hm-2（N0）,N 90 kg·hm-2（N1）,N 180 kg·hm-2（N2）and N 270 kg·hm-2（N3）were set to analyze the effects of nitrogen fertilizer on proso millet filling characteristics,yield and quality,and to clarify the physiological mechanisms of nitrogen fertilizer in regulating qualities by taking into account the structure and properties of proso millet starch and protein.Through the transcriptome and metabolome technologies,the molecular mechanism of nitrogen fertilizer in regulating the quality of proso millet was revealed,and the synergistic enhancement of yield and quality of proso millet was ultimately achieved through the cultivation measures of nitrogen application.The main research results were as follows:（1）Nitrogen application affected grain appearance quality and pasting properties of proso millet by influencing soluble starch synthase（SS）,starch branching enzyme（SBE）,granule-bound starch synthase（GBSS）activities and structure of proso millet flour.Nitrogen application significantly increased the activities of NR and GS but decreased the GBSS activity,resulting in higher protein content and lower amylose content,L and b values.Nitrogen application increased the SS activity and disordered structure in waxy variety（W139）,resulting in structural disorganization,increase in setback viscosity（SB）and a decrease in breakdown viscosity（BD）and pasting temperature（PT）.However,nitrogen application increased peak viscosity（PV）,BD and SB of non-waxy variety（N297）by increasing SBE activity and decreasing hydrophobic functional group content.Yield of proso millet increased significantly with increasing N level,but NPFP and ANUE of proso millet decreased.NPFP,ANUE and yield（waxy variety W139）of proso millet were highest at the N1 level.（2）Nitrogen fertilizer affected the mode of starch and protein binding and secondary structure during cooking leading to differences in cooking characteristics;it affected the proso millet congee by regulating the ester metabolite.During the cooking process,the proso millet flour after nitrogen application mainly showed smooth lumpy material and high proportion ofα-helix structure,which reduced the water absorption and swelling ratio of proso millet grains during the cooking process,and increased the in vitro digestibility of starch,thus affecting the homogeneity and texture of the congee.The aroma of proso millet congee was mainly fruity,floral and burnt with a slight woody-earthiness,whereas nitrogen fertilizer affected the three main substances of esters,terpenes and aldehydes which in turn led to the differences in aroma.Compared to non-waxy variety（N297）,the waxy variety（W139）had higher starch crystallinity,but lower contents of amylose,unsaturated fatty acid,which contributed to the high digestibility and low iodine-blue values of the cooked proso millet,thus requiring longer cooking time,high volatile metabolite（r OAV）values,and a more intense aroma.（3）Nitrogen fertilizer had a significant effect on the amino acid content and secondary structure of proso millet protein,leading to changes in physicochemical properties.Nitrogen application significantly increased essential amino acid content but loosened the dense structure of protein,which presented larger particles.Nitrogen fertilizer did not change the composition of proso millet protein subunits,but decreased the accumulation of subunits at 20k Da and 54 k Da.In addition,nitrogen treatment increased the content of hydrophobic amino acids and theβsheet ratio,which improved the oil absorption capacity,emulsification characteristics and thermal stability of protein.The balanced amino acid ratio of proso millet protein can be supplemented to other cereals as a nutritional supplement,and also had good application prospects in the food industry due to its better functional properties（especially the waxy variety W139）.（4）Nitrogen fertilizer affected the pasting and rheological properties of starch from proso millet grains.As nitrogen levels increased,the starch surface became uneven,the number of small granules increased and the structure of the starch granules shifted towards greater complexity.Nitrogen fertilizer does not change the starch crystallization type,but increases the relative crystallinity and ordered structure of starch,contributing to the stability of the starch structure and helping to increase the enthalpy of pasting（ΔH）.Furthermore,nitrogen treatment also significantly increased the PV,BD,SB and final viscosities（FV）,which deteriorated the shear resistance and storage capacity of proso millet starch-based foods during processing and transportation.Rheological analysis showed that proso millet starch exhibited typical characteristics of the pseudoplastic fluid.After applying nitrogen fertilizer,starch gels of proso millet had good prospects for application in 3D printing products due to strong resistance to shear thinning,gel strength and yield stress.（5）Transcriptome analysis revealed that the regulation of nitrogen fertilizer on proso millet grains mainly occurred at the 20 d（S2）-30 d（S3）after flowering.Nitrogen application up-regulated glutamine synthetase（GS）and glutamate decarboxylase（GAD）genes to promote glutamate synthesis;meanwhile,it down-regulated differentially expressed genes of phenylalanine,tyrosine and tryptophan biosynthesis,and glycolysis/gluconeogenesis metabolic pathways.Nitrogen fertilization inhibited the expression of GBSS and SS genes and promoted the expression of 1,4-α-glucan branching enzyme（GBE）gene,which reduced the amylose content and activated the synthesis of amylopectin.The expression ofβ-furanofructokinase（sacA）gene,hexokinase（HK）gene,and sucrose synthase（SuS）gene of the waxy variety（W139）variety was significantly lower than that of the non-waxy variety（N297）,which led to the starch content of waxy variety（W139）being lower than that of the non-waxy variety（N297）.（6）Differential metabolites regulated by nitrogen fertilizer were identified by metabolomic analysis to be mainly involved in lipid metabolism,amino acid metabolism and carbohydrate metabolism pathways.Nitrogen application inhibited sugar synthesis and accumulation in the grains,leading to impeded starch synthesis and accumulation;however,it promoted the TCA cycle,increasing energy supply and carbon skeleton utilization.In addition,nitrogen fertilizer promoted tryptophan accumulation and improved the nutritional quality by regulating amino acid metabolic pathways.It also maintained stress signal transduction and supported protein biosynthesis in endosperm by regulating hormone levels.The accumulation of most lipid metabolites under nitrogen treatment maintained cellular structure and metabolic energy storage under stress.In summary,nitrogen application promoted alanine,aspartate and glutamate metabolism and TCA cycle,inhibited glycolysis/gluconeogenesis pathways,resulting in protein accumulation and blocked starch synthesis.The GBSS and SS genes were down-regulated while the GBE gene was up-regulated after nitrogen application,which decreased the amylose content and activated the synthesis of amylopectin.Nitrogen fertilization increased relative crystallinity and ordered structure and affected the rheological properties and pasting properties.Meanwhile,nitrogen application increased the hydrophobic amino acid content andβsheet ratio of protein,leading to changes in protein solubility and stability.Nitrogen fertilizer increased the yield but reduced the appearance quality and cooking quality of proso millet grains. |
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