Manfei Li

Flavonoid 3-hydroxylase (F3H) is a key enzyme in the flavonoid biosynthetic pathway; however, its functional roles in maize responses to abiotic stress remain poorly understood. In this study, 14 maize F3H proteins (ZmF3Hs) were identified and characterized through bioinformatics analyses, expression profiling, and functional validation. The ZmF3H genes exhibited structural and physicochemical diversity and were unevenly distributed across seven maize chromosomes. Phylogenetic and synteny analyses classified the ZmF3Hs into four subfamilies and revealed strong evolutionary conservation with homologs from rice, wheat, and sorghum. Conserved motif and promoter analyses suggested functional divergence and multilayered regulatory control. Subcellular localization prediction indicated that ZmF3H6 is localized in the cytoplasm, which was further confirmed by transient expression assays in Nicotiana benthamiana. Expression profiling demonstrated that ZmF3Hs are responsive to multiple abiotic stresses, including cold, heat, and waterlogging. Functional analysis using virus-induced gene silencing revealed that suppression of ZmF3H6 significantly reduced flavonol accumulation and antioxidant enzyme activities while increasing malondialdehyde (MDA) levels under both normal and stress conditions. These findings provide new insights into the biological functions of ZmF3Hs and identify ZmF3H6 as a key regulator of flavonoid biosynthesis and abiotic stress tolerance, offering a promising target for improving stress resilience and flavonoid-mediated defense mechanisms in maize.

The KERNEL NUMBER PER ROW6 (KNR6)-mediated phosphorylation of an adenosine diphosphate ribosylation factor (Arf) GTPase-activating protein (AGAP) forms a key regulatory module for the numbers of spikelets and kernels in the ear inflorescences of maize (Zea mays L.). However, the action mechanism of the KNR6–AGAP module remains poorly understood. Here, we characterized the AGAP-recruited complex and its roles in maize cellular physiology and agronomically important traits. AGAP and its two interacting Arf GTPase1 (ARF1) members preferentially localized to the Golgi apparatus. The loss-of-function AGAP mutant produced by CRISPR/Cas9 resulted in defective Golgi apparatus with thin and compact cisternae, together with delayed internalization and repressed vesicle agglomeration, leading to defective inflorescences and roots, and dwarfed plants with small leaves. The weak agap mutant was phenotypically similar to knr6, showing short ears with fewer kernels. AGAP interacted with KNR6, and a double mutant produced shorter inflorescence meristems and mature ears than the single agap and knr6 mutants. We hypothesized that the coordinated KNR6–AGAP–ARF1 complex modulates vegetative and reproductive traits by participating in vesicle trafficking in maize. Our findings provide a novel mechanistic insight into the regulation of inflorescence development, and ear length and kernel number, in maize.

Increasing grain yield of maize (Zea maysL.) is required to meet the rapidly expanding demands for maize-derived food, feed, and fuel. Breeders have enhanced grain productivity of maize hybrids by pyramiding desirable characteristics for larger ears. However, loci selected for improving grain productivity remain largely unclear. Here, we show that a serine/threonine protein kinase encoding geneKERNEL NUMBER PER ROW6 (KNR6)determines pistillate floret number and ear length. Overexpression ofKNR6or introgression of alleles lacking the insertions of two transposable elements in the regulatory region ofKNR6can significantly enhance grain yield. Further in vitro evidences indicate that KNR6 can interact with an Arf GTPase-activating protein (AGAP) and its phosphorylation by KNR6 may affect ear length and kernel number. This finding provides knowledge basis to enhance maize hybrids grain yield.

The differentiation and subsequent development of plant tissues or organs are tightly regulated at multiple levels, including the transcriptional, posttranscriptional, translational, and posttranslational levels. Transcriptomes define many of the tissue-specific gene expression patterns in maize, and some key genes and their regulatory networks have been established at the transcriptional level. In this study, the sequential window acquisition of all theoretical spectra-mass spectrometry technique was employed as a quantitative proteome assay of four representative maize tissues, and a set of high confidence proteins were identified. Integrated analysis of the proteome and transcriptome revealed that protein abundance was positively correlated with mRNA level with weak to moderate correlation coefficients, but the abundance of key proteins for function or architecture in a given tissue was closely tempo-spatially regulated at the transcription level. A subset of differentially expressed proteins, specifically tissue specific highly expressed proteins, were identified, e.g., reproductive structure and flower development-related proteins in tassel and ear, lipid and fatty acid biosynthetic process-related proteins in immature embryo, and inorganic substance and oxidation reduction responsive proteins in root, potentially revealing the physiology, morphology and function of each tissue. Furthermore, we found many new proteins in specific tissues that were highly correlated with their mRNA levels, in addition to known key factors. These proteome data provide new perspective for understanding many aspects of maize developmental biology. Raw proteomics data are available via ProteomeXchange with identifier PXD008464.