Insights into starch synthesis and amino acid composition of common buckwheat in response to phosphate fertilizer management strategies.

To investigate the response and the regulatory mechanism of common buckwheat starch, amylose, and amylopectin biosynthesis to P management strategies, field experiments were conducted in 2021 and 2022 using three phosphorus (P) levels. Results revealed that the application of 75 kg hm-2 phosphate fertilizer significantly enhanced amylopectin and total starch content in common buckwheat, leading to improved grain weight and starch yield, and decreased starch granule size. The number of upregulated differentially expressed proteins induced by phosphate fertilizer increased with the application rate, with 56 proteins identified as shared differential proteins between different P levels, primarily associated with carbohydrate and amino acid metabolism. Phosphate fertilizer inhibited amylose synthesis by downregulating granule-bound starch synthase protein expression and promoted amylopectin accumulation by upregulating 1,4-alpha-glucan branching enzyme and starch synthase proteins expression. Additionally, Phosphate fertilizer primarily promoted the accumulation of hydrophobic and essential amino acids. These findings elucidate the mechanism of P-induced starch accumulation and offer insights into phosphate fertilizer management and high-quality cultivation of common buckwheat.

Genetic diversity of grain yield traits and identification of a grain weight gene SiTGW6 in foxtail millet.

KEY MESSAGE: Agronomic traits were evaluated in 1250 foxtail millet accessions, and a crucial gene SiTGW6 governing grain yield was identified. Elite haplotypes and dCAPS markers developed for SiTGW6 facilitate molecular breeding. A comprehensive evaluation of phenotypic characteristics and genetic diversity in germplasm resources are important for gene discovery and breeding improvements. In this study, we conducted a comprehensive evaluation of 1250 foxtail millet varieties, assessing seven grain yield-related traits and fourteen common agronomic traits over two years. Principal component analysis, correlation analysis, and cluster analysis revealed a strong positive correlation between 1000-grain weight and grain width with grain yield, emphasizing their importance in foxtail millet breeding. Additionally, we found that panicle weight positively correlated with 1000-grain weight but negatively correlated with branch and tiller numbers, indicating selection factors during domestication and breeding. Using this information, we identified 27 germplasm resources suitable for high-yield foxtail millet breeding. Furthermore, through an integration of haplotype variations and phenotype association analysis, we pinpointed a crucial gene, SiTGW6, responsible for governing grain yield in foxtail millet. SiTGW6 encodes an IAA-glucose hydrolase, primarily localized in the cytoplasm and predominantly expressed in flowering panicles. Employing RNAseq analysis, we identified 1439 differentially expressed genes across various SiTGW6 haplotypes. Functional enrichment analysis indicating that SiTGW6 regulates grain yield through the orchestration of auxin and glucan metabolism, as well as plant hormone signaling pathways. Additionally, we have identified elite haplotypes and developed dCAPS markers for SiTGW6, providing valuable technical tools to facilitate molecular breeding efforts in foxtail millet.

Lipidomics studies reveal dynamic changes in polar lipids of developing endosperm of oat and wheat varieties with differing oil contents.

Polar lipids have biosynthetic pathways which intersect and overlap with triacylglycerol biosynthesis; however, polar lipids have not been well characterized in the developing endosperms of oat with high oil accumulation. The polar lipids in endosperms of oat and wheat varieties having different oil contents were analyzed and compared at different developmental stages. Our study shows that the relative contents of polar lipid by mass were decreased more slowly in wheat than in oat. Phosphatidylcholine and phosphatidylethanolamine were the major phospholipids, which showed similar abundance and gradual decreases during endosperm development in oat and wheat, while lysophospholipids were noticeably higher in oat. Monogalactosyldiacylglycerol showed a gradual increase in wheat and a decrease in oat during endosperm development. The relative contents of some polar lipid species and their unsaturation index were significantly different in their endosperms. These characteristics of polar lipids might indicate an adaption of oat to accommodate oil accumulation.

Gluten-starch microstructure analysis revealed the improvement mechanism of Triticeae on broomcorn millet (Panicum miliaceum L.).

Understanding the mechanism by which Triticeae improves the quality of broomcorn millet (BM) is key to expanding the use of this crop to address food crises and food security. This study aimed to explore the effects of Triticeae on the disulfide bonds, secondary structures, microstructure, and rheological properties of BM dough, and to investigate the potential food applications of BM. Gluten protein, intermolecular SS, and ß-Sheets content of the reconstituted doughs were significantly improved compared with BM dough, which improved disorderly accumulation of starch and gluten-starch interaction in BM dough. CLSM analysis showed that broomcorn millet-common wheat (BM-CW) and broomcorn millet-durum wheat (BM-DW) also possessed larger protein areas, smaller lacunarities, and better gluten-starch interactions in the reconstituted doughs. Disulfide bonds were positively correlated with the gluten network structure, and more disulfide bonds were formed in BM-CW (3.86 µmol/g), which promoted stronger mechanical resistance in BM-CW. Therefore, the combination of BM flour with CW and DW flours had better dough elasticity and stability. Finally, a potential evaluation and optimization scheme for BM as a cooked wheaten food is proposed to improve the reference for future food security and dietary structure adjustment of residents.

Comparative Multi-Omics Analysis of Broomcorn Millet in Response to Anthracocystis destruens Infection.

Anthracocystis destruens is the causal agent of broomcorn millet (Panicum miliaceum) smut disease, which results in serious yield losses in broomcorn millet production. However, the molecular basis underlying broomcorn millet defense against A. destruens is less understood. In this study, we investigated how broomcorn millet responds to infection by A. destruens by employing a comprehensive multi-omics approach. We examined the responses of broomcorn millet across transcriptome, metabolome, and microbiome levels. Infected leaves exhibited an upregulation of genes related to photosynthesis, accompanied by a higher accumulation of photosynthesis-related compounds and alterations in hormonal levels. However, broomcorn millet genes involved in immune response were downregulated post A. destruens infection, suggesting that A. destruens may suppress broomcorn millet immunity. In addition, we show that the immune suppression and altered host metabolism induced by A. destruens have no significant effect on the microbial community structure of broomcorn millet leaf, thus providing a new perspective for understanding the tripartite interaction between plant, pathogen, and microbiota.

Effects of nitrogen fertilizer on the physicochemical, structural, functional, thermal, and rheological properties of mung bean (Vigna radiata) protein.

Nitrogen fertilizer can affect the seed quality of mung bean. However, the effects of nitrogen fertilizer on the properties of mung bean protein (MBP) remain unclear. We investigated the effects of four nitrogen fertilization levels on the physicochemical, structural, functional, thermal, and rheological properties of MBP. The results showed that the amino acid and protein contents of mung bean flour were maximized under 90 kg ha-1 of applied nitrogen treatment. Nitrogen fertilization can alter the secondary and tertiary structure of MBP. The main manifestations are an increase in the proportion of ß-sheet, the exposure of more chromophores and hydrophobic groups, and the formation of loose porous aggregates. These changes improved the solubility, oil absorption capacity, emulsion activity, and foaming stability of MBP. Meanwhile, Thermodynamic and rheological analyses showed that the thermal stability, apparent viscosity, and gel elasticity of MBP were all increased under nitrogen fertilizer treatment. Correlation analysis showed that protein properties are closely related to changes in structure. In conclusion, nitrogen fertilization can improve the protein properties of MBP by modulating the structure of protein molecules. This study provides a theoretical basis for the optimization of mung bean cultivation and the further development of high-quality mung bean protein foods.

Nitrogen regulates the synthesis of hydrophobic amino acids to improve protein structural and gel properties in common buckwheat.

Nitrogen (N) fertilizer impacts the grain quality of common buckwheat, but the effects and regulatory mechanisms of N on various protein parameters of buckwheat are not fully understood. The purpose of this study was to investigate the particle morphology, structural and gel properties, and regulation mechanism of buckwheat protein under four N levels. The bulk density, surface hydrophobicity, particle size, and thermal properties of the buckwheat protein were maximized through the optimal N application (180 kg N/ha), further enhancing the thermal stability of the protein. N application increased the ß-sheet content and reduced the random coil content. Appropriate N fertilizer input enhanced the tertiary structure stability and gel elasticity of buckwheat protein by promoting hydrophobic interactions, disulfide bonds, ionic bonds, storage modulus and loss modulus. The differentially expressed proteins induced by N are primarily enriched in small ribosomal subunit and ribosome, improving protein quality mainly by promoting the synthesis of hydrophobic amino acids. Future agriculture should pay attention to the hydrophobic amino acid content of buckwheat to effectively improve protein quality. This study further advances the application of buckwheat protein in the field of food processing and provides a theoretical basis for the extensive development and utilization of buckwheat protein.

Comparison of the fine structure and physicochemical properties of proso millet (Panicum miliaceum L.) starch from different ecological regions.

Field experiments were conducted to evaluate the morphology, granule size, fine structure, thermal properties, and pasting properties of starch from a waxy (139) and a non-waxy (297) varieties of proso millet grown in Yulin (YY) and Yangling (YL). Compared with the starches from the two varieties grown in YY, the starches from the two varieties grown in YL exhibited higher relative crystallinities, 1045/1022 cm-1 ratio, and amounts of amylopectin long branch chains (APL) but lower 1022/995 cm -1 ratio, amounts of amylopectin short branch chains (APs), and APs/APL ratios. Starches from YL also synthesized long branch-chain amylopectin to enhance intermolecular interactions and form a stable granular structure, which resulted in increased starch gelatinization temperature, enhanced shear resistance, and reduced setback viscosity. Starch from the waxy (139) variety has good application prospects in the food industry because of its high gelatinization temperature and light transmittance and low setback value, which can be ascribed to its extremely low amylose content, polydispersity index, high molecular weight, and dispersed molecular density. It may serve as a reference for applying proso millet starches in the food industry and developing breeding programs to improve starch quality.

Mapping of major QTL and candidate gene analysis for hull colour in foxtail millet (Setaria italica (L.) P. Beauv.).

BACKGROUND: Hull colour is an important morphological marker for selection in seed production of foxtail millet. However, the molecular mechanisms underlying hull colour variation remain unknown. RESULTS: An F7 recombinant inbred line (RIL) population containing 215 lines derived from Hongjiugu × Yugu18 was used to analyze inheritance and detect the quantitative trait loci (QTL) for four hull colour traits using major gene plus polygene mixed inheritance analysis and composite interval mapping (CIM) in four environments. Genetic analysis revealed that the hull colour L* value (HCL*) was controlled by two major genes plus additive polygenes, the hull colour a* value (HCa*) was controlled by three major genes, the hull colour b* value (HCb*) was controlled by two major genes plus polygenes, and the hull colour C* value (HCC*) was controlled by four major genes. A high-density genetic linkage map covering 1227.383 cM of the foxtail millet genome, with an average interval of 0.879 cM between adjacent bin markers, was constructed using 1420 bin markers. Based on the genetic linkage map and the phenotypic data, a total of 39 QTL were detected for these four hull colour traits across four environments, each explaining 1.50%-49.20% of the phenotypic variation. Of these, six environmentally stable major QTL were co-localized to regions on chromosomes 1 and 9, playing a major role in hull colour. There were 556 annotated genes within the two QTL regions. Based on the functions of homologous genes in Arabidopsis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) gene annotations, five genes were predicted as candidate genes for further studies. CONCLUSIONS: This is the first study to use an inheritance model and QTL mapping to determine the genetic mechanisms of hull colour trait in foxtail millet. We identified six major environmentally stable QTL and predicted five potential candidate genes to be associated with hull colour. These results advance the current understanding of the genetic mechanisms underlying hull colour traits in foxtail millet and provide additional resources for application in genomics-assisted breeding and potential isolation and functional characterization of the candidate genes.

Effects of mulching types on the yield and water utilization by broomcorn millet (Panicum miliaceum): Results of a study in the Loess Plateau, China.

Mulching practices have been widely adopted to improve rainfed crop productivity. However, the major resources including water, heat, and light that influenced the yield of broomcorn millet in different dryland regions have rarely been explored. A three-season field experiment with three mulching practices i.e. traditional planting with non-mulching (TP), ridge-furrow mulching system (RF), and plastic film mulching (PFM) was conducted in three semi-arid regions in the Loess Plateau, China, i.e. Guyuan city (GY), Huining county (HN), and Yulin city (YL) between 2020 and 2022 to investigate the impacts of mulching regimes on soil hydrothermal conditions, agronomic characteristics, leaf photosynthetic properties, broomcorn millet yield, and water use efficiency (WUE). Results showed that both PFM and RF treatments increased soil temperature and moisture, and enhanced dry matter accumulation by promoting leaf photosynthetic capacity and chlorophyll content, thereby improving broomcorn millet yield and WUE. PFM and RF increased the average broomcorn millet yield by 15.08% and 24.86% at GY site, 20.86% and 25.61% at HN site, and 15.75% and 25.57% at YL site, respectively, and increased the average WUE by 16.31% and 27.48% at GY site, 23.21% and 28.80% at HN site, 15.55% and 28.57% at YL site, respectively. Partial least squares path modeling analysis revealed that soil moisture was an important environmental factor in determining broomcorn millet yield. Overall, RF practice can be taken to improve the management of agricultural climate factors and maximize yield, thereby promoting the sustainable development of dryland agriculture in the Loess Plateau.

Biochar and Nitrogen Fertilizer Change the Quality of Waxy and Non-Waxy Broomcorn Millet (Panicum miliaceum L.) Starch.

The overuse of nitrogen fertilizers has led to environmental pollution, which has prompted the widespread adoption of biochar as a soil conditioner in agricultural production. To date, there has been a lack of research on the effects of biochar and its combination with nitrogen fertilizer on the quality of broomcorn millet (Panicum miliaceum L.) starch. Thus, this study examined the physicochemical characteristics of starch in two types of broomcorn millet (waxy and non-waxy) under four different conditions, including a control group (N0), nitrogen fertilizer treatment alone (N150), biochar treatment alone (N0+B), and a combination of biochar and nitrogen fertilizer treatments (N150+B). The results showed that, in comparison to the control, all the treatments, particularly N150+B, decreased the content of amylose and gelatinization temperature and enhanced the starch transparency gel consistency and swelling power. In addition, biochar can improve the water solubility of starch and the gelatinization enthalpy. Importantly, the combination of biochar and nitrogen fertilizer increased the proportion of A-granules, final viscosity, starch content, and the average degree of amylopectin in polymerization. Thus, this research indicates that the combinations of biochar and nitrogen fertilizer result in the most significant improvement in the quality of starch produced from broomcorn millet.

A graph-based genome and pan-genome variation of the model plant Setaria.

Setaria italica (foxtail millet), a founder crop of East Asian agriculture, is a model plant for C4 photosynthesis and developing approaches to adaptive breeding across multiple climates. Here we established the Setaria pan-genome by assembling 110 representative genomes from a worldwide collection. The pan-genome is composed of 73,528 gene families, of which 23.8%, 42.9%, 29.4% and 3.9% are core, soft core, dispensable and private genes, respectively; 202,884 nonredundant structural variants were also detected. The characterization of pan-genomic variants suggests their importance during foxtail millet domestication and improvement, as exemplified by the identification of the yield gene SiGW3, where a 366-bp presence/absence promoter variant accompanies gene expression variation. We developed a graph-based genome and performed large-scale genetic studies for 68 traits across 13 environments, identifying potential genes for millet improvement at different geographic sites. These can be used in marker-assisted breeding, genomic selection and genome editing to accelerate crop improvement under different climatic conditions.

Widely targeted metabolomic analysis revealed the effects of alkaline stress on nonvolatile and volatile metabolites in broomcorn millet grains.

Broomcorn millet (BM) is a future smart food. However, no information is available on the metabolism of BM grains under alkaline stress. In this study, the effects of alkaline stress on nonvolatile and volatile metabolites in the BM grains of two varieties (S223 and T289) were investigated through metabolomics approaches. All 933 nonvolatile metabolites and 313 volatile metabolites were identified, with 114 and 89 nonvolatile metabolites and 16 and 20 volatile metabolites accumulating differentially under normal vs. alkaline stresses of S223 and T289, respectively. The results indicated that alkaline stress altered phenylpropanoids, flavonoids, flavone and flavonol, valine, leucine, and isoleucine biosynthesis, as well as arginine, proline, tryptophan, and ascorbate metabolism. The effects of alkaline stress were not identical between the two varieties, which could lead to variations in active substance content. These results provide valuable information for further studies on food chemistry and the functional food development of BM grains.

Linkages of microbial community structure and root exudates: Evidence from microbial nitrogen limitation in soils of crop families.

Rhizosphere microorganisms are critical for crop nutrient cycling and soil ecological functions in agroecosystem soils; however, there is limited information regarding the role of root exudates in determining soil microbial communities and functions in plant-soil systems, especially for microbial nutrient limitations. In the present study, rhizosphere soil samples were collected from the main food crop families, including maize, soybean, potato, and buckwheat, representing the cereals, Leguminosae, Solanaceae, and Polygonaceae families, in the northern Loess Plateau, China, to investigate soil microbial co-occurrences and assembly processes and the relationship between soil microbes and root exudates. The results showed that the crop families greatly regulated the soil microbial community composition and assembly, and all microorganisms of the four species were subjected to N limitation via the vector analysis. The topological properties of the soil microbial networks varied with the crop family, demonstrating that the ecological relationships of bacterial taxa are more complex than those of fungi. Stochastic processes were more important in stimulating assembly across the four crop families; the non-dominated process governed >60 % of the critical ecological turnover in community assembly, whereas dispersal limitation was the key factor influencing fungal community assembly. Furthermore, the metabolic profiles of root exudates in response to microbial N limitation varied by family. Microbial function and metabolic limitations were strongly associated with variations in root exudates, especially amino acids and organic acids, which were directly facilitated by crop families. Our results highlight the key roles of root exudates in stimulating microbial community structure and ecological functions from the perspective of microbial nutrient limitation and improve our understanding of plant-microbe interactions in agricultural ecosystems.

Phosphorous fertilization alleviates shading stress by regulating leaf photosynthesis and the antioxidant system in mung bean (Vigna radiata L.).

Shading can limit photosynthesis and plant growth. Understanding how phosphorus (P) application mitigates the effects of shading stress on morphology and physiology of mung beans (Vigna radiata L.) is of great significance for the establishment of efficient planting structures and optimizing P-use management. The effects of various light environments (non-shading stress, S0; low light stress, S1; severe shading stress, S2) on the growth of two mung bean cultivars (Xilv1 and Yulv1) and the role of P application (0 kg ha-1, P0; 90 kg ha-1, P1; 150 kg ha-1, P2) in such responses were investigated in a field experiment. Our results demonstrated that shading decreased the dry matter accumulation of mung bean markedly by limiting photosynthesis capacity and disrupting agronomic traits. For the leaf areas of the two cultivars, chlorophyll a+b, the net photosynthetic and electron transport rates were increased by 16.8%, 20.0%, 15.5%, and 12.5% under P1 treatment, and by 32.4%, 40.3%, 16.3% and 12.8% under P2 treatment, respectively, when compared to those for the non-fertilized plants under shading stress. These responses resulted in increased light capture and weak light utilization. Moreover, the activities of superoxide dismutase and peroxidase were enhanced by 20.9% and 43.7%, respectively; malondialdehyde and superoxide anion contents were reduced by 18.6% and 14.1%, respectively, under P application. These findings suggest that P application moderately mitigates the damage caused by shading stress and enhances tolerance by regulating mung bean growth. In addition, Xilv1 was more sensitive to P under shading stress than Yulv1.

Biogeographic Pattern and Network of Rhizosphere Fungal and Bacterial Communities in Panicum miliaceum Fields: Roles of Abundant and Rare Taxa.

Unraveling how microbial interactions and assembly process regulate the rhizosphere abundant and rare taxa is crucial for determining how species diversity affects rhizosphere microbiological functions. We assessed the rare and abundant taxa of rhizosphere fungal and bacterial communities in proso millet agroecosystems to explore their biogeographic patterns and co-occurrence patterns based on a regional scale. The taxonomic composition was significantly distinct between the fungal and bacterial abundant and rare taxa. Additionally, the rare taxa of bacteria and fungi exhibited higher diversity and stronger phylogenetic clustering than those of the abundant ones. The phylogenetic turnover rate of abundant taxa of bacteria was smaller than that of rare ones, whereas that of fungi had the opposite trend. Environmental variables, particularly mean annual temperature (MAT) and soil pH, were the crucial factors of community structure in the rare and abundant taxa. Furthermore, a deterministic process was relatively more important in governing the assembly of abundant and rare taxa. Our network analysis suggested that rare taxa of fungi and bacteria were located at the core of maintaining ecosystem functions. Interestingly, MAT and pH were also the important drivers controlling the main modules of abundant and rare taxa. Altogether, these observations revealed that rare and abundant taxa of fungal and bacterial communities showed obvious differences in biogeographic distribution, which were based on the dynamic interactions between assembly processes and co-occurrence networks.

Nitrogen fertilizer affects starch synthesis to define non-waxy and waxy proso millet quality.

Understanding the effect of nitrogen fertilization on the quality of proso millet is key to expanding the use of this crop to address water scarcity and food security. Therefore, this study determined the impact of nitrogen fertilization on the proso millet quality. Nitrogen fertilization significantly increased the NR and GS activities and decreased the GBSSase activity, resulting in an increase in protein content and reduction in amylose content and L*, which decreased the appearance quality. Nitrogen fertilization increased the proportion of short amylopectin chains, resulting in a more disordered carbohydrate structure, and decreased the proportion of hydrophilic functional groups, contributing to an increase in setback viscosity and decrease in pasting temperature in the waxy (w139) variety. In contrast, the non-waxy (n297) variety exhibited a larger proportion of long amylopectin chains, lower ordered structure and hydrophobic functional groups after nitrogen fertilization, which strengthened the inter- and intramolecular forces of starch colloids.

Transcriptome analysis reveals new insights in the starch biosynthesis of non-waxy and waxy broomcorn millet (Panicum miliaceum L.).

Broomcorn millet is a popular cereal with health benefits, and its grains are rich in starch. However, the differences in the pathway and key genes involved in starch biosynthesis of waxy and non-waxy broomcorn millet grain remain unclear. Therefore, the grain and starch physicochemical index and transcriptomic analyses of two genotypes of broomcorn millet were conducted at 3, 6, 9, 12, 15, 18, and 21 days after pollination. The phenotypic and physiological results indicated that the starch synthetic process of non-waxy and waxy broomcorn millet was significantly different. The amylose, amylopectin, and total starch contents of non-waxy broomcorn millet were 1.99, 4.74, and 6.73 mg/grain, while those of waxy broomcorn millet were 0.34, 5.94, and 6.28 mg/grain, respectively. The transcriptomic analysis revealed that 106 differentially expressed genes were identified, which were mainly enriched in the "amino sugar and nucleotide sugar metabolism", "pyruvate metabolism", "galactose metabolism", and "starch and sucrose metabolism" pathways. The WGCNA suggested that a total of 31 hub genes were correlated with starch biosynthesis. These findings provide a new approach to studying the starch synthesis in broomcorn millet.

Cadmium tolerance and accumulation from the perspective of metal ion absorption and root exudates in broomcorn millet.

Cadmium (Cd) is a persistent heavy metal that poses environmental and public health concerns. This study aimed to identify the potential biomarkers responsible for Cd tolerance and accumulation by investigating the response of the content of essential metal elements, transporter gene expression, and root exudates to Cd stress in broomcorn millet (Panicum miliaceum). A hydroponics experiment was conducted using two broomcorn millet cultivars with distinct Cd tolerance levels and accumulation phenotypes (Cd-tolerant and Cd-sensitive cultivars). Cd stress inhibited lateral root growth, especially in the Cd-sensitive cultivar. Furthermore, Cd accumulation was significantly greater in the Cd-tolerant cultivar than in the Cd-sensitive cultivar. Cd stress significantly inhibited the absorption of essential metal elements and significantly increased the calcium concentration. Differentially expressed genes involved in metal ion transport were identified via transcriptome analysis. Cd stress altered the composition of root exudates, thus increasing lipid species and decreasing alkaloid, lignan, sugar, and alcohol species. Moreover, Cd stress significantly reduced most alkaloid, organic acid, and phenolic acid exudates in the Cd-tolerant cultivar, while it increased most lipid and phenolic acid exudates in the Cd-sensitive cultivar. Some significantly changed root exudates (ferulic acid, O-coumaric acid, and spermine) are involved in the phenylalanine biosynthesis, and arginine and proline metabolic pathways, thus, may be potential biomarkers of Cd stress response. Overall, metal ion absorption and root exudates are critical for Cd tolerance and accumulation in broomcorn millet. These findings provide valuable insights into improving Cd phytoremediation by applying mineral elements or metabolites.

Structure, physicochemical, functional and in vitro digestibility properties of non-waxy and waxy proso millet starches.

The structural, physicochemical, gel textural, rheological, and in vitro digestibility properties as well as their relationships of non-waxy proso millet starch (NPMS) and waxy proso millet starch (WPMS) were evaluated by taking normal corn starch (CS) and potato starch (PS) as controls. Proso millet starch was mostly polygonal or spherical, with an A-type crystalline structure. Proso millet starch contained more short-branched chains (DP 6-24) compared with CS and PS. WPMS possessed higher crystallinity and more short-range ordered structures than NPMS. NPMS displayed higher pasting temperature, retrogradation rate and shear thinning degree, and lower gelatinization temperature and enthalpy than WPMS. The hardness and chewiness of starch gel formed by NPMS were higher than those of WPMS. All starch samples exhibited shear thinning behavior in the steady-flow test and typical elastic solid behavior in the dynamic rheological test. Moreover, NPMS was considered a potential formula for functional foods, with its lower rapidly digestible starch (RDS) and higher resistant starch (RS) contents than WPMS, CS, and PS. This paper revealed the influence of amylose content and structure on the physicochemical properties of different proso millet starch.