Scientia Agricultura Sinica ›› 2025, Vol. 58 ›› Issue (9): 1702-1718.doi: 10.3864/j.issn.0578-1752.2025.09.003

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Integrated Multi-Omics Elucidates the Pigmentation Dynamics During Post-Harvest Maturation in Foxtail Millet (Setaria italica)

ZHANG YiRu1(), HAN Xue1(), YAO XinJie3, FENG Jun1, WEI AiLi1, LI WenChao1, ZHANG Bin2, HAN YuanHuai2, LI HongYing2   

  1. 1 College of Biological Sciences and Technology, Taiyuan Normal University, Yuci 030619, Shanxi
    2 College of Agriculture, Shanxi Agricultural University, Taigu 030801, Shanxi
    3 College of Agriculture, Ningxia University, Yinchuan 750021
  • Received:2024-10-14 Accepted:2024-12-12 Online:2025-05-08 Published:2025-05-08

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Abstract:

【Objective】 The aim of the study is to investigate the variation characteristics of starch and carotenoid content in grains of the foxtail millet during post-harvest maturation, and to elucidate the molecular mechanism of this phenomenon. The study will expand new insights into carotenoid metabolism in grains, thereby providing theoretical support for the stabilization of millet color, deep processing of millet, and storage. 【Method】 The carotenoid and starch content in grains from ten varieties of foxtail millet were measured after storage for 0, 30, 60, and 90 days, respectively. Changes in starch granules of representative materials during the four storage stages were observed using confocal laser scanning microscopy. Transcriptome and proteome analyses were conducted to investigate differential genes and proteins related to starch and carotenoid metabolic pathways at different storage stages for the same material. 【Result】 Among the 10 foxtail millet varieties, the carotenoid content in the millet mainly showed an upward trend from 0 to 60 days of post-harvest maturation and began to decline after 90 days, but remained higher than that of 0 days. The starch content showed an upward trend after 30 days of post-harvest maturation and then declined overall. Microscopic observation revealed a gradual increase in carotenoid fluorescence in millet starch granules after 30 days of post-harvest maturation, with a tendency for starch plastids to convert into chromoplasts. Furthermore, the transcriptome and proteome analyses identified 1 344 differentially expressed genes (DEGs) and 224 differentially expressed proteins (DEPs), which were mainly enriched in starch degradation and carotenoid synthesis pathways. A metabolic network regulation was also constructed.【Conclusion】 During of post-harvest maturation of the foxtail millet, there is an increase trend in the carotenoid content and a decrease trend in the starch content of the millet. It is speculated that starch degradation leads to an increase in pyruvate content, which enters the plastid methylerythritol phosphate (MEP) pathway to promote carotenoid synthesis. Towards the end of post-harvest maturation of the foxtail millet, the downstream synthesis of abscisic acid is inhibited, resulting in the accumulation of substances such as beta-carotene and zeaxanthin, which subsequently affect changes in the millet color.

Key words: foxtail millet, post-harvest maturation, millet color, starch, carotenoids, transcriptomics, proteomics

Fig. 1

Standard curve for starch"

Table 1

List of primers used for qRT-PCR analysis"

基因ID Gene ID 正向引物Forward primer (5′-3′) 反向引物Reverse primer (5′-3′)
Seita.6G181400 GCTACGCCTACATCCTAACCC CCGATCTTCGTGATGACTTTGT
Seita.6G181600 TACAACAGCCTCAATGCCCA GTAACCTTGGGGTGAGACGG
Seita.1G331000 ACAAGGTCATGCAGGGCTAC AGGCCCCAGTCGAAGAAATG
Seita.9G229700 GTACTACAACACCCGGCACC CATCTCGACGCAGGTGAAGT
Seita.4G263300 CAGTACACCCAGCTCATCGG AGCAACGTTCTTGTACCCGT
Seita.5G134800 TTCTGTTCATCCCATCTTCCTG CCCTCCTTACAATTTCGCCTA
Seita.8G083400 GCCAAGGATGAACGGAACCT GAGCTCCATGTTCTCCCCAC
Seita.3G059500 CGTCGCACTCAGGAAAGTGT GGCAGAACCGTCGAGCTTAT
Seita.9G239800 TGGAGAAGTGGTTGTGGTGC TCAAGAGCGAAAGTGCATGG
Seita.4G108300 CCGGTCATATTGGTCTGCGA TAAGCTGGGCTCTTGGATGC
actin TGCTCAGTGGAGGCTCAACA CCAGACACTGTACTTGCGCTC

Fig. 2

The change characteristic of carotenoid (A) and starch (B) contents in grains of foxtail millet during the storage period JG21: Jingu 21; CN35: Changnong 35; GS4: Gongsui 4; XYG: Xiangyanggu; MS: Maosu; YG11: Yugu 11; JG11: Jigu 11; YG1: Yugu 1; QH2: Qinhuang 2; JF52: Jinfen 52"

Fig. 3

The change characteristic of carotenoid (A) and starch (B) contents in grains of xiaomi during the storage period Different letter represents significant differences at the 5% level between different groups, respectively"

Fig. 4

Microscopic observations of millet plastids during the after-ripening period of the foxtail millet Black particles: Starch granules; Red circles: The green fluorescence of carotenoids in starch granules"

Table 2

The information of transcriptome sequencing data"

样品
Sample		 原始数据
Raw reads		 高质量数据
Clean reads		 总比对率
Total mapped (%)		 过滤后数据
Clean bases (G)		 Q20
(%)		 Q30
(%)		 GC
(%)
0d-1 58375418 55633756 97.22 8.35 98.33 95.17 57.54
0d-2 58769864 55823920 97.21 8.37 98.48 95.64 57.53
0d-3 56265292 53767766 97.19 8.07 98.41 95.40 57.68
90d-1 53164314 51169888 96.36 7.68 98.08 94.82 56.38
90d-2 53275414 50375952 96.26 7.56 98.37 95.33 56.72
90d-3 44176254 41831696 96.36 6.27 98.30 95.17 57.02

Fig. 5

Analysis of GO enrichment (A) and KEGG enrichment (B) of differentially expressed genes during after-repening period of xiaomi"

Table 3

Proteome identification results"

数据库蛋白质数目
Database		 鉴定肽段总数
Peptides		 鉴定蛋白质总数
Identified proteins		 定量蛋白质总数
Quantification proteins
46514 64191 8182 8182

Fig. 6

Analysis of differential protein expression for 0 d and 90 d after millet maturation with GO enrichment (A) and KEGG enrichment (B)"

Fig. 7

Combined transcriptome and proteome analysis of 0 day and 90 day in the millet of xiaomi during after-ripening period A: Venn map for DEGs and DEPs; B: Bar map for DEGs and DEPs; C: Gene Ontology (GO) categories for DEGs and DEPs; D: KEGG enrichment analysis for DEGs and DEPs"

Fig. 8

Network regulation map of starch degradation and carotenoid synthesis pathways A: Metabolic pathways of starch degradation and carotenoid synthesis; B: Heatmap of gene expression related to these pathways; C: Heatmap of protein expression related to these pathways. Boxes represent enzymes, green indicates downregulation of expression, red indicates upregulation, and blue indicates no significant difference in expression levels. AMY: α-amylase; BMY: β-amylase; MGAM: Maltose enzyme-glucoamylase; HK: Hexokinase; PGM: Phosphoglucomutase; GPI: Glucose-6-phosphate isomerase; SPP: Sucrose phosphate synthase; SPS: Sucrose phosphate synthase; INA: Sucrase enzyme; FRK: Fructokinase; PFP: Adenosine diphosphate-dependent phosphofructokinase; FBA: Fructose-1,6-bisphosphate aldolase; PGK: Glycerol phosphate kinase; PGAM: 2,3-diphosphoglycerate-dependent phosphoglycerate mutase; ENO: Enolase; PK: Pyruvate Kinase; PDH: Pyruvate dehydrogenase; AACT: Acetyl-CoA acetyltransferase; DXP: Deoxymannose-6-phosphate synthase; DXR: Deoxy-mannose-6-phosphate reductase isomerase; PSY: Bicyclic lycopene synthase; PDS: Phytoene desaturase; Z-ISO: ζ-Carotene Isomerase; ZDS: Carotene dehydrogenase; CRTISO, Carotenoid isomerase; LCYB, β-Lycopene cyclase; LCYE, ε-Lycopene cyclase; BCH, β-carotene hydroxylase; ZEP: Zeaxanthin epoxidase; NCED: 9-cis-epoxycarotenoid dioxygenase; ABA2: Flavoxate dehydrogenase; AAO3: Abscisic acid aldehyde oxidase"

Fig. 9

qRT-PCR verification of gene expression (A) with RNA-Seq expression patterns (B) *: Significance at P<0.05; **: Significance at P<0.01; ***: Significance at P<0.001"

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