Discovering of the transcriptional regulatory network involved in starch biosynthesis in sorghum grains

doi:10.1016/j.pld.2025.11.006

Abstract

Abstract: Starch is the most abundant accumulated substance in the grains of sorghum (Sorghum bicolor), the 5th most world-widely cultivated cereal crop, and is widely used by humans, especially in the direction of brewing in China. However, there are currently few reports on the starch biosynthesis regulatory mechanism in sorghum grains. Here, we employed RNA-seq and ATAC-seq strategies to discover the transcriptional regulation network responsible for starch biosynthesis in sorghum grains. Our results profiled all mRNAs in the sorghum grains at nine development stages covering the inflorescence, and grains from 3 to 30 days after pollination (DAP). Analysis of the gene sets determined temporal programs of gene expression, including thousands of transcription factor (TF) genes. We found a close correlation between the sequentially expressed gene sets and distinct cellular and metabolic programs of the developing grains. The cis-elements serving as binding sites of multiple TFs were identified via a comparative ATAC-seq assay. Cis-elements capable of binding TFs were also identified within the promoter regions of starch biosynthesis related genes (SBRGs). Moreover, the NAC family TF of SbNAC68 highly expressed in developing grains and demonstrated co-expression patterns with SBRGs. Furthermore, SbNAC68 was confirmed to bind to 5'-ACGCAA-3', a typical motif of binding site for the TFs from NAC family, to affect the promotor activities of SBRGs and regulate their transcriptions. Collectively, through multiply omics strategies and the case dissection of SbNAC68, the present study provides molecular insights of transcriptional regulations into starch biosynthesis in sorghum grains.

Key words: Sorghum (Sorghum bicolor), Starch biosynthesis, Transcriptional regulation, SbNAC68

摘要： Starch is the most abundant accumulated substance in the grains of sorghum (Sorghum bicolor), the 5th most world-widely cultivated cereal crop, and is widely used by humans, especially in the direction of brewing in China. However, there are currently few reports on the starch biosynthesis regulatory mechanism in sorghum grains. Here, we employed RNA-seq and ATAC-seq strategies to discover the transcriptional regulation network responsible for starch biosynthesis in sorghum grains. Our results profiled all mRNAs in the sorghum grains at nine development stages covering the inflorescence, and grains from 3 to 30 days after pollination (DAP). Analysis of the gene sets determined temporal programs of gene expression, including thousands of transcription factor (TF) genes. We found a close correlation between the sequentially expressed gene sets and distinct cellular and metabolic programs of the developing grains. The cis-elements serving as binding sites of multiple TFs were identified via a comparative ATAC-seq assay. Cis-elements capable of binding TFs were also identified within the promoter regions of starch biosynthesis related genes (SBRGs). Moreover, the NAC family TF of SbNAC68 highly expressed in developing grains and demonstrated co-expression patterns with SBRGs. Furthermore, SbNAC68 was confirmed to bind to 5'-ACGCAA-3', a typical motif of binding site for the TFs from NAC family, to affect the promotor activities of SBRGs and regulate their transcriptions. Collectively, through multiply omics strategies and the case dissection of SbNAC68, the present study provides molecular insights of transcriptional regulations into starch biosynthesis in sorghum grains.

关键词: Sorghum (Sorghum bicolor), Starch biosynthesis, Transcriptional regulation, SbNAC68

Zhizhai Liu, Xiangling Gong, Jing Li, Hong Duan, Tingting Dai, Wei Wei, Min Yin, Yi Zheng, Hameed Gul, Jiuguang Wang, Chaoxian Liu, Qianlin Xiao. Discovering of the transcriptional regulatory network involved in starch biosynthesis in sorghum grains[J]. Plant Diversity, 2026, 48(03): 544-556.

References

[1] Ahmed, Z., Tetlow, I.J., Ahmed, R., et al., 2015. Protein-protein interactions among enzymes of starch biosynthesis in high-amylose barley genotypes reveal differential roles of heteromeric enzyme complexes in the synthesis of A and B granules. Plant Sci. 233, 95-106.
[2] Anders, S., Pyl, P.T., Huber, W., 2015. HTSeq-a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166-169.
[3] Baguma, Y., Sun, C., Borén, M., et al., 2008. Sugar-mediated semidian oscillation of gene expression in the cassava storage root regulates starch synthesis. Plant Signal. Behav. 3, 439-445.
[4] Bahaji, A., Li, J., Sánchez-López, Á.M., et al., 2014. Starch biosynthesis, its regulation and biotechnological approaches to improve crop yields. Biotechnol. Adv. 32, 87-106.
[5] Bajic, M., Maher, K.A., Deal, R.B., 2018. Identification of open chromatin regions in plant genomes using ATAC-Seq. Methods Mol. Biol. 1675, 183-201.
[6] Bello, B.K., Hou, Y., Zhao, J., et al., 2019. NF-YB1-YC12-bHLH144 complex directly activates Wx to regulate grain quality in rice (Oryza sativa L.). Plant Biotechnol. J. 17, 1222-1235.
[7] Blauth, S.L., Yao, Y., Klucinec, J.D., et al., 2001. Identification of Mutator insertional mutants of Starch-Branching Enzyme 2a in corn. Plant Physiol. 125, 1396-1405.
[8] Boehlein, S.K., Shaw, J.R., Stewart, J.D., et al., 2009. Studies of the kinetic mechanism of maize endosperm ADP-glucose pyrophosphorylase uncovered complex regulatory properties. Plant Physiol. 152, 1056-1064.
[9] Buléon, A., Colonna, P., Planchot, V., et al., 1998. Starch granules: structure and biosynthesis. Int. J. Biol. Macromol. 23, 85-112.
[10] Burton, R.A., Jenner, H., Carrangis, L., et al., 2002. Starch granule initiation and growth are altered in barley mutants that lack isoamylase activity. Plant J. 31, 97-112.
[11] Cao, R., Zhao, S., Jiao, G., et al., 2022. OPAQUE3, encoding a transmembrane bZIP transcription factor, regulates endosperm storage protein and starch biosynthesis in rice. Plant Commun. 3, 100463.
[12] Chen, E., Yu, H., He, J., et al., 2023. The transcription factors ZmNAC128 and ZmNAC130 coordinate with Opaque2 to promote endosperm filling in maize. Plant Cell 35, 4066-4090.
[13] Chen, J., Huang, B., Li, Y., et al., 2011. Synergistic influence of sucrose and abscisic acid on the genes involved in starch synthesis in maize endosperm. Carbohydr. Res. 346, 1684-1691.
[14] Chen, J., Yi, Q., Cao, Y., et al., 2016. ZmbZIP91 regulates expression of starch synthesis-related genes by binding to ACTCAT elements in their promoters. J. Exp. Bot. 67, 1327-1338.
[15] Chen, S., Zhou, Y., Chen, Y., et al., 2018. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34, i884-i890.
[16] Consortium, T.G.O., 2019. The Gene ontology resource: 20 years and still GOing strong. Nucleic Acids Res. 47, D330-D338.
[17] Delrue, B., Fontaine, T., Routier, F., et al., 1992. Waxy Chlamydomonas reinhardtii: monocellular algal mutants defective in amylose biosynthesis and granule-bound starch synthase activity accumulate a structurally modified amylopectin. J. Bacteriol. 174, 3612-3620.
[18] Ernst, J., Bar-Joseph, Z., 2006. STEM: a tool for the analysis of short time series gene expression data. BMC Bioinformatics 7, 191.
[19] Fu, F., Xue, H., 2010. Coexpression analysis identifies rice starch Regulator1, a rice AP2/EREBP family transcription factor, as a novel rice starch biosynthesis regulator. Plant Physiol. 154, 927-938.
[20] Guan, J., Wang, Z., Liu, S., et al., 2022. Transcriptome analysis of developing wheat grains at rapid expanding phase reveals dynamic gene expression patterns. Biology 11, 281.
[21] Grimaud, F., James, M.G., Myers, A.M., 2008. Proteome and phosphoproteome analysis of starch granule-associated proteins from normal maize and mutants affected in starch biosynthesis. J. Exp. Bot. 59, 3395-3406.
[22] Hirose, T., Terao, T., 2004. A comprehensive expression analysis of the starch synthase gene family in rice (Oryza sativa L.). Planta 220, 9-16.
[23] Hu, Y., Li, Y., Weng, J., et al., 2021. Coordinated regulation of starch synthesis in maize endosperm by microRNAs and DNA methylation. Plant J. 105, 108-123.
[24] Hu, Y., Li, Y., Zhang, J., et al., 2012. Binding of ABI4 to a CACCG motif mediates the ABA-induced expression of the ZmSSI gene in maize (Zea mays L.) endosperm. J. Exp. Bot. 63, 5979-5989.
[25] Huang, H., Xie, S., Xiao, Q., et al., 2016. Sucrose and ABA regulate starch biosynthesis in maize through a novel transcription factor, ZmEREB156. Sci. Rep. 6, 27590.
[26] Huang, L., Tan, H., Zhang, C., et al., 2021. Starch biosynthesis in cereal endosperms: an updated review over the last decade. Plant Commun. 2, 100237.
[27] James, M.G., Denyer, K., Myers, A.M., 2003. Starch synthesis in the cereal endosperm. Curr. Opin. Plant Biol. 6, 215-222.
[28] Jeon, J.S., Ryoo, N., Hahn, T.R., et al., 2010. Starch biosynthesis in cereal endosperm. Plant Physiol. Biochem. 48, 383-392.
[29] Jin, S.K., Xu, L.N., Leng, Y.J., et al., 2023. The OsNAC24-OsNAP protein complex activates OsGBSSI and OsSBEI expression to fine-tune starch biosynthesis in rice endosperm. Plant Biotechnol. J. 21, 2224-2240.
[30] Kötting, O., Kossmann, J., Zeeman, S.C., et al., 2010. Regulation of starch metabolism: the age of enlightenment? Curr. Opin. Plant Biol. 13, 321-329.
[31] Kanehisa, M., Araki, M., Goto, S., et al., 2007. KEGG for linking genomes to life and the environment. Nucleic Acids Res. 36, D480-D484.
[32] Kang, X., Gao, W., Cui, B., et al., 2023. Structure and genetic regulation of starch formation in sorghum (Sorghum bicolor (L.) Moench) endosperm: a review. Int. J. Biol. Macromol. 239, 124315.
[33] Kawakatsu, T., Yamamoto, M.P., Touno, S.M., et al., 2009. Compensation and interaction between RISBZ1 and RPBF during grain filling in rice. Plant J. 59, 908-920.
[34] Keeling, P.L., Myers, A.M., 2010. Biochemistry and genetics of starch synthesis. Annu. Rev. Food Sci. Technol. 1, 271-303.
[35] Kim, D., Langmead, B., Salzberg, S.L., 2015. HISAT: a fast spliced aligner with low memory requirements. Nat. Methods 12, 357-360.
[36] Kolbe, A., Tiessen, A., Schluepmann, H., et al., 2005. Trehalose 6-phosphate regulates starch synthesis via posttranslational redox activation of ADP-glucose pyrophosphorylase. Proc. Natl. Acad. Sci. U.S.A. 102, 11118-11123.
[37] Lü, B., Guo, Z., Liang, J., 2008. Effects of the activities of key enzymes involved in starch biosynthesis on the fine structure of amylopectin in developing rice (Oryza sativa L.) endosperms. Sci. China C Life Sci. 51, 863-871.
[38] Li, H., Gilbert, R.G., 2018. Starch molecular structure: the basis for an improved understanding of cooked rice texture. Carbohydr. Polym. 195, 9-17.
[39] Liu, B., Meng, S., Yang, J., et al., 2025. Carbohydrate flow during grain filling: phytohormonal regulation and genetic control in rice (Oryza sativa). J. Integr. Plant Biol. 67, 1086-1104.
[40] Liu, Y., Hou, J., Wang, X., et al., 2020. The NAC transcription factor NAC019-A1 is a negative regulator of starch synthesis in wheat developing endosperm. J. Exp. Bot. 71, 5794-5807.
[41] Liu, Y., Xi, W., Wang, X., et al., 2023. TabHLH95-TaNF-YB1 module promotes grain starch synthesis in bread wheat. J. Genet. Genomics 50, 883-894.
[42] Liu, Z., Jiang, S., Jiang, L., et al., 2022. Transcription factor OsSGL is a regulator of starch synthesis and grain quality in rice. J. Exp. Bot. 73, 3417-3430.
[43] Love, M.I., Huber, W., Anders, S., 2014. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550.
[44] Ma, B., Zhang, L., He, Z., 2023. Understanding the regulation of cereal grain filling: the way forward. J. Integr. Plant Biol. 65, 526-547.
[45] Makhmoudova, A., Williams, D., Brewer, D., et al., 2014. Identification of multiple phosphorylation sites on maize endosperm starch branching enzyme IIb, a key enzyme in amylopectin biosynthesis. J. Biol. Chem. 289, 9233-9246.
[46] Nakamura, Y., Ono, M., Utsumi, C., et al., 2012. Functional interaction between plastidial starch phosphorylase and starch branching enzymes from rice during the synthesis of branched maltodextrins. Plant Cell Physiol. 53, 869-878.
[47] Ohdan, T., Francisco, P.J.T., Hirose, T., et al., 2005. Expression profiling of genes involved in starch synthesis in sink and source organs of rice. J. Exp. Bot. 56, 3229-3244.
[48] Pontieri, P., Troisi, J., Calcagnile, M., et al., 2022. Chemical composition, fatty acid and mineral content of food-grade white, red and black sorghum varieties grown in the mediterranean environment. Foods 11, 436.
[49] Qi, X., Li, S., Zhu, Y., et al., 2016. ZmDof3, a maize endosperm-specific Dof protein gene, regulates starch accumulation and aleurone development in maize endosperm. Plant Mol. Biol. 93, 7-20.
[50] Radchuk, V.V., Borisjuk, L., Sreenivasulu, N., et al., 2009. Spatiotemporal profiling of starch biosynthesis and degradation in the developing barley grain. Plant Physiol. 150, 190-204.
[51] Rakshit, S., Hariprasanna, K., Gomashe, S., et al., 2014. Changes in area, yield gains, and yield stability of sorghum in major sorghum-producing countries, 1970 to 2009. Crop Sci. 54, 1571-1584.
[52] Ren, Y., Huang, Z., Jiang, H., et al., 2021. A heat stress responsive NAC transcription factor heterodimer plays key roles in rice grain filling. J. Exp. Bot. 72, 2947-2964.
[53] Roberts, A., Trapnell, C., Donaghey, J., et al., 2011. Improving RNA-Seq expression estimates by correcting for fragment bias. Genome Biol. 12, R22.
[54] Seung, D., Smith, A.M., 2019. Starch granule initiation and morphogenesis-progress in Arabidopsis and cereals. J. Exp. Bot. 70, 771-784.
[55] Shannon, J.C., Pien, F.M., Cao, H., et al., 1998. Brittle-1, an adenylate translocator, facilitates transfer of extraplastidial synthesized ADP-glucose into amyloplasts of maize endosperms. Plant Physiol. 117, 1235-1252.
[56] Song, Y., Luo, G., Shen, L., et al., 2020. TubZIP28, a novel bZIP family transcription factor from Triticum urartu, and TabZIP28, its homologue from Triticum aestivum, enhance starch synthesis in wheat. New Phytol. 226, 1384-1398.
[57] Stamova, B.S., Laudencia-Chingcuanco, D., Beckles, D.M., 2009. Transcriptomic analysis of starch biosynthesis in the developing grain of hexaploid wheat. Int. J. Plant Genom. 2009, 1-23.
[58] Subramanian, A., Tamayo, P., Mootha, V.K., et al., 2005. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. U.S.A. 102, 15545-15550.
[59] Tetlow, I.J., Beisel, K.G., Cameron, S., et al., 2008. Analysis of protein complexes in wheat amyloplasts reveals functional interactions among starch biosynthetic enzymes. Plant Physiol. 146, 1878-1891.
[60] Tetlow, I.J., Wait, R., Lu, Z., et al., 2004. Protein phosphorylation in amyloplasts regulates starch branching enzyme activity and protein-protein interactions. Plant Cell 16, 694-708.
[61] Tickle, P., Burrell, M.M., Coates, S.A., et al., 2009. Characterization of plastidial starch phosphorylase in Triticum aestivum L. endosperm. J. Plant Physiol. 166, 1465-1478.
[62] Walley, J.W., Shen, Z., Sartor, R., et al., 2013. Reconstruction of protein networks from an atlas of maize seed proteotypes. Proc. Natl. Acad. Sci. U.S.A. 110, E4808-E4817.
[63] Wang, J., Chen, Z., Zhang, Q., et al., 2020. The NAC transcription factors OsNAC20 and OsNAC26 regulate starch and storage protein synthesis. Plant Physiol. 184, 1775-1791.
[64] Wang, J., Xu, H., Zhu, Y., et al., 2013. OsbZIP58, a basic leucine zipper transcription factor, regulates starch biosynthesis in rice endosperm. J. Exp. Bot. 64, 3453-3466.
[65] Wang, X., Liu, Y., Hao, C., et al., 2023. Wheat NAC-A18 regulates grain starch and storage proteins synthesis and affects grain weight. Theor. Appl. Genet. 136, 123.
[66] Wu, J., Chen, L., Chen, M., et al., 2019. The DOF-domain transcription factor ZmDOF36 positively regulates starch synthesis in transgenic maize. Front. Plant Sci. 10, 465.
[67] Xiao, Q., Huang, T., Cao, W., et al., 2022a. Profiling of transcriptional regulators associated with starch biosynthesis in sorghum (Bicolor sorghum L.). Front. Plant Sci. 13, 999747.
[68] Xiao, Q., Liu, T., Ling, M., et al., 2022b. Genome-wide identification of DOF gene family and the mechanism dissection of SbDof21 regulating starch biosynthesis in sorghum. Int. J. Mol. Sci. 23, 12152.
[69] Xiao, Q., Wang, Y., Du, J., et al., 2017. ZmMYB14 is an important transcription factor involved in the regulation of the activity of the ZmBT1 promoter in starch biosynthesis in maize. FEBS J. 284, 3079-3099.
[70] Xiao, Q., Wang, Y., Li, H., et al., 2021. Transcription factor ZmNAC126 plays an important role in transcriptional regulation of maize starch synthesis-related genes. Crop J. 9, 192-203.
[71] Yu, Y., Zhu, D., Ma, C., et al., 2016. Transcriptome analysis reveals key differentially expressed genes involved in wheat grain development. Crop J. 4, 92-106.
[72] Zeeman, S.C., Kossmann, J., Smith, A.M., 2010. Starch: its metabolism, evolution, and biotechnological modification in plants. Annu. Rev. Plant Biol. 61, 209-234.
[73] Zeeman, S.C., Smith, S.M., Smith, A.M., 2007. The diurnal metabolism of leaf starch. Biochem. J. 401, 13-28.
[74] Zemach, A., Kim, M.Y., Silva, P., et al., 2010. Local DNA hypomethylation activates genes in rice endosperm. Proc. Natl. Acad. Sci. U.S.A. 107, 18729-18734.
[75] Zhang, Z., Dong, J., Ji, C., et al., 2019. NAC-type transcription factors regulate accumulation of starch and protein in maize seeds. Proc. Natl. Acad. Sci. U.S.A. 116, 11223-11228.
[76] Zhang, Z., Zheng, X., Yang, J., et al., 2016. Maize endosperm-specific transcription factors O₂ and PBF network the regulation of protein and starch synthesis. Proc. Natl. Acad. Sci. U.S.A. 113, 10842-10847.
[77] Zhu, F., 2014. Structure, physicochemical properties, modifications, and uses of sorghum starch. Compr. Rev. Food Sci. Food Saf. 13, 597-610.
[78] Zhu, Y., Cai, X.L., Wang, Z.Y., et al., 2003. An interaction between a MYC protein and an EREBP protein is involved in transcriptional regulation of the rice Wx gene. J. Biol. Chem. 278, 47803-47811.