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SUMMARY: Maize plastid terminal oxidase1 (ZmPTOX1) plays a pivotal role in seed development by upholding redox balance within seed plastids. This study focuses on characterizing the white kernel mutant 3735 (wk3735) mutant, which yields pale‐yellow seeds characterized by heightened protein but reduced carotenoid levels, along with delayed germination compared to wild‐type (WT) seeds. We successfully cloned and identified the target gene ZmPTOX1, responsible for encoding maize PTOX—a versatile plastoquinol oxidase and redox sensor located in plastid membranes. While PTOX's established role involves regulating redox states and participating in carotenoid metabolism in Arabidopsis leaves and tomato fruits, our investigation marks the first exploration of its function in storage organs lacking a photosynthetic system. Through our research, we validated the existence of plastid‐localized ZmPTOX1, existing as a homomultimer, and established its interaction with ferredoxin‐NADP+ oxidoreductase 1 (ZmFNR1), a crucial component of the electron transport chain (ETC). This interaction contributes to the maintenance of redox equilibrium within plastids. Our findings indicate a propensity for excessive accumulation of reactive oxygen species (ROS) in wk3735 seeds. Beyond its known role in carotenoids' antioxidant properties, ZmPTOX1 also impacts ROS homeostasis owing to its oxidizing function. Altogether, our results underscore the critical involvement of ZmPTOX1 in governing seed development and germination by preserving redox balance within the seed plastids. Significance Statement: Aside from its established role as a vital regulator of redox states and its involvement in carotenoid metabolism, as observed in Arabidopsis leaves and tomato fruits, this study marks the inaugural exploration into PTOX's function within storage organs lacking a photosynthetic system. Our findings elucidate how ZmPTOX1 significantly impacts protein and starch accumulation in seeds, along with the pace of seed germination. Moreover, it actively maintains redox equilibrium across various stages of seed development and germination. [ABSTRACT FROM AUTHOR]

Anthocyanin, a kind of flavonoid, plays a crucial role in plant resistance to abiotic stress. Salt stress is a kind of abiotic stress that can damage the growth and development of plant seedlings. However, limited research has been conducted on the involvement of maize seedlings in salt stress resistance via anthocyanin accumulation, and its potential molecular mechanism is still unclear. Therefore, it is of great significance for the normal growth and development of maize seedlings to explore the potential molecular mechanism of anthocyanin improving salt tolerance of seedlings via transcriptome analysis. In this study, we identified two W22 inbred lines (tolerant line pur–W22 and sensitive line bro–W22) exhibiting differential tolerance to salt stress during seedling growth and development but showing no significant differences in seedling characteristics under non–treatment conditions. In order to identify the specific genes involved in seedlings' salt stress response, we generated two recombinant inbred lines (RILpur–W22 and RILbro–W22) by crossing pur–W22 and bro–W22, and then performed transcriptome analysis on seedlings grown under both non–treatment and salt treatment conditions. A total of 6100 and 5710 differentially expressed genes (DEGs) were identified in RILpur–W22 and RILbro–W22 seedlings, respectively, under salt–stressed conditions when compared to the non–treated groups. Among these DEGs, 3160 were identified as being present in both RILpur–W22 and RILbro–W22, and these served as commonly stressed EDGs that were mainly enriched in the redox process, the monomer metabolic process, catalytic activity, the plasma membrane, and metabolic process regulation. Furthermore, we detected 1728 specific DEGs in the salt–tolerant RILpur–W22 line that were not detected in the salt–sensitive RILbro–W22 line, of which 887 were upregulated and 841 were downregulated. These DEGs are primarily associated with redox processes, biological regulation, and the plasma membrane. Notably, the anthocyanin synthesis related genes in RILpur–W22 were strongly induced under salt treatment conditions, which was consistented with the salt tolerance phenotype of its seedlings. In summary, the results of the transcriptome analysis not only expanded our understanding of the complex molecular mechanism of anthocyanin in improving the salt tolerance of maize seedlings, but also, the DEGs specifically expressed in the salt–tolerant line (RILpur–W22) provided candidate genes for further genetic analysis. [ABSTRACT FROM AUTHOR]

Summary: RUBylation plays essential roles in plant growth and development through regulating Cullin‐RING ubiquitin E3 ligase (CRL) activities and the CRL‐mediated protein degradations. However, the function of RUBylation in regulating kernel development remains unclear.Through genetic and molecular analyses of a small kernel 501 (smk501) mutant in maize (Zea mays), we cloned the smk501 gene, revealed its molecular function, and defined its roles in RUBylation pathway and seed development.Smk501 encodes a RUBylation activating enzyme E1 subunit ZmECR1 (E1 C‐TERMINAL RELATED 1) protein. Destruction in RUBylation by smk501 mutation resulted in less embryo and endosperm cell number and smaller kernel size. The transcriptome and proteome profiling, hormone evaluation and cell proliferation observation revealed that disturbing ZmECR1 expression mainly affects pathways on hormone signal transduction, cell cycle progression and starch accumulation during kernel development. In addition, mutant in zmaxr1 (Auxin resistant 1), another RUB E1 subunit, also showed similar defects in kernel development. Double mutation of zmecr1 and zmaxr1 lead to empty pericarp kernel phenotype.RUBylation is a novel regulatory pathway affecting maize kernel development, majorly through its functions in modifying multiple cellular progresses. [ABSTRACT FROM AUTHOR]

The black layer (BL) is traditionally used as an indicator for kernel harvesting in maize, as it turns visibly dark when the kernel reaches physiological maturity. However, the molecular roles of BL in kernel development have not been fully elucidated. In this work, microscopy images showed that BL began to appear at a growth stage earlier than 10 days after pollination (DAP), and its color gradually deepened to become dark as the development period progressed. Scanning electron microscopy observations revealed that BL is a tissue structure composed of several layers of cells that are gradually squeezed and compressed during kernel development. Laser-capture microdissection (LCM) was used to sample BL and its neighboring inner tissue, basal endosperm transfer layer (BETL), and outer tissue, inner epidermis (IEP), from 20 DAP of kernels. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry profiling (MALDI-TOF MS profiling) detected 41, 104, and 120 proteins from LCM-sampled BL, BETL, and IEP, respectively. Gene ontology (GO) analysis indicated that the 41 BL proteins were primarily involved in the response to stress and stimuli. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis found that the BL proteins were enriched in several defense pathways, such as the ascorbate and aldarate metabolic pathways. Among the 41 BL proteins, six were BL-specific proteins that were only detected from BL. Annotations of five BL-specific proteins were related to stress responses. During kernel development, transcriptional expression of most BL proteins showed an increase, followed by a decrease, and reached a maximum zero to 20 DAP. These results suggest a role for BL in stress responses for protecting filial tissue against threats from maternal sides, which helps to elucidate the biological functions of BL. [ABSTRACT FROM AUTHOR]