Identification of transcription factors regulating starch biosynthesis in maize through integrated GWAS and transcriptomic analysis

Kernel starch content (SC) is a major contributor to maize grain yield and end-use quality, but its genetic improvement is limited by incomplete understanding of genes linking natural variation to starch accumulation during endosperm development. Here, we aimed to identify starch-associated transcription factors (TFs) with breeding potential by integrating multi-environment phenotyping, genome-wide association studies (GWAS), transcriptome profiling of SC-contrasting subpopulations, haplotype analysis, and functional characterisation of key candidate regulators. Kernel SC showed broad variation across four environments, ranging from 56.33% to 75.35%, with significant correlations among environments (r = 0.27–0.56, P < 0.01). Lines from the BSSS subpopulation exhibited consistently higher SC than those from the PA subpopulation, with increasing of 0.82%–2.79%. Comparative transcriptomic analysis of developing endosperm identified significant expression differences in starch biosynthesis genes, including 18 genes downregulated (0.41–0.90-fold) and four genes upregulated (1.23–1.74-fold) in BSSS. Several transcription factors (TFs) identified by GWAS showed coordinated expression with starch biosynthesis genes; seven of these were differentially expressed between BSSS and PA subpopulations and significantly correlated with multiple starch biosynthesis genes. Haplotype analysis showed that favourable Hap1 haplotypes of ZmMYB71, ZmMYB4, and ZmGNAT16 were enriched in BSSS and were associated with a 1.51%–3.30% increase in kernel SC. Functional assays revealed that ZmMYB71 acts as a negative regulator: its overexpression suppressed key starch biosynthesis genes, Sh1, Sh2, and GBSSI, reduced AGPase, GBSS, and SSS activities by 3.26%–21.39%, and decreased kernel SC by 1.70%–4.91%. Conversely, ZmMYB71 loss-of-function mutants showed upregulation of starch biosynthesis genes, increased enzyme activities (5.79%–17.20%), and increased kernel SC (2.67%–5.92%). This study identifies major transcriptional regulators underlying natural variation in maize kernel SC by integrating multi-environment phenotyping, GWAS, transcriptome profiling, and haplotype analysis. ZmMYB71, ZmMYB4, and ZmGNAT16 were prioritized as key candidate TFs associated with SC variation, and functional validation confirmed ZmMYB71 as a regulator of starch accumulation. Favourable haplotypes of these TFs were associated with increased kernel SC, providing practical targets for marker-assisted selection and genetic improvement of starch content in maize.