ADP-Glc pyrophosphorylase (AGPase), which catalyzes the transformation of ATP and glucose-1-phosphate (Glc-1-P) into adenosine diphosphate glucose (ADP-Glc), acts as a rate-limiting enzyme in crop starch biosynthesis. Prior research has hinted at the regulation of AGPase by phosphorylation in maize. However, the identification and functional implications of these sites remain to be elucidated. In this study, we identified the phosphorylation site (serine at the 31st position of the linear amino acid sequence) of the AGPase large subunit (Sh2) using iTRAQ^TM. Subsequently, to ascertain the impact of Sh2 phosphorylation on AGPase, we carried out site-directed mutations creating Sh2-S31A (serine residue replaced with alanine) to mimic dephosphorylation and Sh2-S31D (serine residue replaced with aspartic acid) or Sh2-S31E (serine residue replaced with glutamic acid) to mimic phosphorylation. Preliminary investigations were performed to determine Sh2 subcellular localization, its interaction with Bt2, and the resultant AGPase enzymatic activity. Our findings indicate that phosphorylation exerts no impact on the stability or localization of Sh2. Furthermore, none of these mutations at the S31 site of Sh2 seem to affect its interaction with Bt2 (smaller subunit). Intriguingly, all S31 mutations in Sh2 appear to enhance AGPase activity when co-transfected with Bt2, with Sh2-S31E demonstrating a substantial five-fold increase in AGPase activity compared to Sh2. These novel insights lay a foundational groundwork for targeted improvements in AGPase activity, thus potentially accelerating the production of ADP-Glc (the primary substrate for starch synthesis), promising implications for improved starch biosynthesis, and holding the potential to significantly impact agricultural practices.

Brassinosteroids are a recently discovered group of substances that promote plant growth and productivity. Photosynthesis, which is vital for plant growth and high productivity, is strongly influenced by brassinosteroid signaling. However, the molecular mechanism underlying the photosynthetic response to brassinosteroid signaling in maize remains obscure. Here, we performed integrated transcriptome, proteome, and phosphoproteomic analyses to identify the key photosynthesis pathway that responds to brassinosteroid signaling. Transcriptome analysis suggested that photosynthesis antenna proteins and carotenoid biosynthesis, plant hormone signal transduction, and MAPK signaling in CK VS EBR and CK VS Brz were significantly enriched in the list of differentially expressed genes upon brassinosteroids treatment. Consistently, proteome and phosphoproteomic analyses indicated that photosynthesis antenna and photosynthesis proteins were significantly enriched in the list of differentially expressed proteins. Thus, transcriptome, proteome, and phosphoproteome analyses showed that major genes and proteins related to photosynthesis antenna proteins were upregulated by brassinosteroids treatment in a dose-dependent manner. Meanwhile, 42 and 186 transcription factor (TF) responses to brassinosteroid signals in maize leaves were identified in the CK VS EBR and CK VS Brz groups, respectively. Our study provides valuable information for a better understanding of the molecular mechanism underlying the photosynthetic response to brassinosteroid signaling in maize.

Maize is a main food and feed crop with great production potential and high economic benefits. Improving its photosynthesis efficiency is crucial for increasing yield. Maize photosynthesis occurs mainly through the C4 pathway, and NADP-ME (NADP-malic enzyme) is a key enzyme in the photosynthetic carbon assimilation pathway of C4 plants. ZmC4-NADP-ME catalyzes the release of CO₂ from oxaloacetate into the Calvin cycle in the maize bundle sheath. Brassinosteroid (BL) can improve photosynthesis; however, its molecular mechanism of action remains unclear. In this study, transcriptome sequencing of maize seedlings treated with epi-brassinolide (EBL) showed that differentially expressed genes (DEGs) were significantly enriched in photosynthetic antenna proteins, porphyrin and chlorophyll metabolism, and photosynthesis pathways. The DEGs of C4-NADP-ME and pyruvate phosphate dikinase in the C4 pathway were significantly enriched in EBL treatment. Co-expression analysis showed that the transcription level of ZmNF-YC2 and ZmbHLH157 transcription factors was increased under EBL treatment and moderately positively correlated with ZmC4-NADP-ME. Transient overexpression of protoplasts revealed that ZmNF-YC2 and ZmbHLH157 activate C4-NADP-ME promoters. Further experiments showed ZmNF-YC2 and ZmbHLH157 transcription factor binding sites on the −1616 bp and −1118 bp ZmC4 NADP-ME promoter. ZmNF-YC2 and ZmbHLH157 were screened as candidate transcription factors mediating brassinosteroid hormone regulation of the ZmC4 NADP-ME gene. The results provide a theoretical basis for improving maize yield using BR hormones.

Photosynthesis, which is the foundation of crop growth and development, is accompanied by complex transcriptional regulatory mechanisms. Research has established that brassinosteroids (BRs) play a role in regulating plant photosynthesis, with the majority of research focusing on the physiological level and regulation of rate-limiting enzymes in the dark reactions of photosynthesis. However, studies on their effects on maize photosynthesis, specifically on light-harvesting antenna proteins, have yet to be conducted. The peripheral light-harvesting antenna protein Lhcb5 is crucial for capturing and dissipating light energy. Herein, by analyzing the transcriptomic data of maize seedling leaves treated with 24-epibrassinolide (EBR) and verifying them using qPCR experiments, we found that the MYBR17 transcription factor may regulate the expression of the photosynthetic light-harvesting antenna protein gene. Further experiments using protoplast transient expression and yeast one-hybrid tests showed that the maize transcription factor MYBR17 responds to EBR signals and binds to the promoter of the light-harvesting antenna protein Lhcb5, thereby upregulating its expression. These results were validated using an Arabidopsis mybr17 mutant. Our results offer a theoretical foundation for the application of BRs to enhance the photosynthetic efficiency of maize.

Plastid division regulatory genes play a crucial role in the morphogenesis of chloroplasts and amyloplasts. Chloroplasts are the main sites for photosynthesis and metabolic reactions, while amyloplasts are the organelles responsible for forming and storing starch granules. The proper division of chloroplasts and amyloplasts is essential for plant growth and yield maintenance. Therefore, this study aimed to examine the J175 (FtsZ2-2) gene, cloned from an ethyl methanesulphonate (EMS) mutant involved in chloroplast and amyloplast division in maize, through map-based cloning. We found that J175 encodes a cell division protein, FtsZ (filamentous temperature-sensitive Z). The FtsZ family of proteins is widely distributed in plants and may be related to the division of chloroplasts and amyloplasts. The J175 protein is localized in plastids, and its gene is expressed across various tissues. From the seedling stage, the leaves of the j175 mutant exhibited white stripes, while the division of chloroplasts was inhibited, leading to a significant increase in volume and a reduction in their number. Measurement of the photosynthetic rate showed a significant decrease in the photosynthetic efficiency of j175. Additionally, the division of amyloplasts in j175 grains at different stages was impeded, resulting in irregular polygonal starch granules. RNA-seq analyses of leaves and kernels also showed that multiple genes affecting plastid division, such as FtsZ1, ARC3, ARC6, PDV1-1, PDV2, and MinE1, were significantly downregulated. This study demonstrates that the maize gene j175 is essential for maintaining the division of chloroplasts and amyloplasts and ensuring normal plant growth, and provides an important gene resource for the molecular breeding of maize.