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Cohesin complexes maintain sister chromatid cohesion to ensure proper chromosome segregation during mitosis and meiosis. In plants, the exact components and functions of the cohesin complex remain poorly understood. Here, we positionally cloned the classic maize (Zea mays) mutant defective kernel 15 (dek15), revealing that it encodes a homolog of S...
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... dek15 kernels could be clearly distinguished from the wild type by their lighter color as early as 12 DAP, and the immature mutant kernels were smaller than the wild type at all developmental stages (Supplemental Figure 1C). The dek15 embryos appeared to be more severely affected than the endosperm (Figures 1G to 1I; Supplemental Figure 2); while the wild type formed the typical embryonic structures, including the scutellum, leaf primordia, shoot apical meristem, and root apical meristem, the dek15 embryos arrested during early development, and were much smaller than those of the wild type ( Figure 1H). In addition there was no relationship between defective embryo size and endosperm size in the dek15 kernels at 15 DAP (Supplemental Figure 2B). ...
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... dek15 embryos appeared to be more severely affected than the endosperm (Figures 1G to 1I; Supplemental Figure 2); while the wild type formed the typical embryonic structures, including the scutellum, leaf primordia, shoot apical meristem, and root apical meristem, the dek15 embryos arrested during early development, and were much smaller than those of the wild type ( Figure 1H). In addition there was no relationship between defective embryo size and endosperm size in the dek15 kernels at 15 DAP (Supplemental Figure 2B). ...
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... electron microscopy observations revealed that the immature dek15 endosperm contained fewer and smaller starch grains and protein bodies than that of the wild type ( Figure 2A). In the mature dek15 endosperm, scanning electron microscopy observation revealed that the starch grains were also smaller at this stage, and that the mature endosperm contained less of the proteinaceous matrix than the wild type ( Figure 2B). ...
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... electron microscopy observations revealed that the immature dek15 endosperm contained fewer and smaller starch grains and protein bodies than that of the wild type ( Figure 2A). In the mature dek15 endosperm, scanning electron microscopy observation revealed that the starch grains were also smaller at this stage, and that the mature endosperm contained less of the proteinaceous matrix than the wild type ( Figure 2B). The starch and protein contents of the dek15 endosperm were sig- nificantly lower than those of the wild-type endosperm, both as a percentage of kernel weight ( Figure 2C) and as the average contents per kernel ( Figure 2D). ...
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... the mature dek15 endosperm, scanning electron microscopy observation revealed that the starch grains were also smaller at this stage, and that the mature endosperm contained less of the proteinaceous matrix than the wild type ( Figure 2B). The starch and protein contents of the dek15 endosperm were sig- nificantly lower than those of the wild-type endosperm, both as a percentage of kernel weight ( Figure 2C) and as the average contents per kernel ( Figure 2D). ...
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... the mature dek15 endosperm, scanning electron microscopy observation revealed that the starch grains were also smaller at this stage, and that the mature endosperm contained less of the proteinaceous matrix than the wild type ( Figure 2B). The starch and protein contents of the dek15 endosperm were sig- nificantly lower than those of the wild-type endosperm, both as a percentage of kernel weight ( Figure 2C) and as the average contents per kernel ( Figure 2D). ...
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... dek15 kernels exhibited a drastic reduction in their total starch and protein contents (Figure 2), which in turn reduced their kernel weights. The decrease in nutrient storage is presumed to result from the functional failure of the endosperm cells. ...
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... decrease in nutrient storage is presumed to result from the functional failure of the endosperm cells. Nutrients are transported from the maternal vascular tissue through the BETL into the endosperm (Gómez et al., 2009); however, the reduced BETL cell wall ingrowth in dek15 was likely an obstacle for nutrition transportation into the developing kernels ( Figures 6C and 6D). In addition, the decreased cell number and abnormal morphology of the dek15 SE cells may also affect grain filling (Figures 6E and 6F; Supplemental Figure 7). ...
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... For transmission electron microscopy analysis, mn7 and WT endosperms at 15 DAP were prepared as previously described (He et al., 2019). The sections were observed with a Hitachi H7600 transmission electron microscope (Japan). ...
The Elongator complex was originally identified as an interactor of hyperphosphorylated RNA polymerase II (RNAPII) in yeast and has histone acetyltransferase (HAT) activity. However, the genome‐wide regulatory roles of Elongator on transcriptional elongation and histone acetylation remain unclear. We characterized a maize miniature seed mutant, mn7 and map‐based cloning revealed that Mn7 encodes one of the subunits of the Elongator complex, ZmELP1. ZmELP1 deficiency causes marked reductions in the kernel size and weight. Molecular analyses showed that ZmELP1 interacts with ZmELP3, which is required for H3K14 acetylation (H3K14ac), and Elongator complex subunits interact with RNA polymerase II (RNAPII) C‐terminal domain (CTD). Genome‐wide analyses indicated that loss of ZmELP1 leads to a significant decrease in the deposition of H3K14ac and the CTD of phosphorylated RNAPII on Ser2 (Ser2P). These chromatin changes positively correlate with global transcriptomic changes. ZmELP1 mutation alters the expression of genes involved in transcriptional regulation and kernel development. We also showed that the decrease of Ser2P depends on the deposition of Elongator complex‐mediated H3K14ac. Taken together, our results reveal an important role of ZmELP1 in the H3K14ac‐dependent transcriptional elongation, which is critical for kernel development.
... Maize is an important crop that serves as a model plant for r esear ch on kernel de v elopment. Although numerous genes regulate kernel de v elopment in maize (22)(23)(24)(25)(26)(27)(28)(29), how RNAPs might contribute to kernel de v elopment is unkno wn. Ho we v er, two recent reports re v ealed the important roles of Pol III in maize kernel de v elopment. ...
Maize (Zea mays) kernel size is an important factor determining grain yield; although numerous genes regulate kernel development, the roles of RNA polymerases in this process are largely unclear. Here, we characterized the defective kernel 701 (dek701) mutant that displays delayed endosperm development but normal vegetative growth and flowering transition, compared to its wild type. We cloned Dek701, which encoded ZmRPABC5b, a common subunit to RNA polymerases I, II and III. Loss-of-function mutation of Dek701 impaired the function of all three RNA polymerases and altered the transcription of genes related to RNA biosynthesis, phytohormone response and starch accumulation. Consistent with this observation, loss-of-function mutation of Dek701 affected cell proliferation and phytohormone homeostasis in maize endosperm. Dek701 was transcriptionally regulated in the endosperm by the transcription factor Opaque2 through binding to the GCN4 motif within the Dek701 promoter, which was subjected to strong artificial selection during maize domestication. Further investigation revealed that DEK701 interacts with the other common RNA polymerase subunit ZmRPABC2. The results of this study provide substantial insight into the Opaque2-ZmRPABC5b transcriptional regulatory network as a central hub for regulating endosperm development in maize.
... Many Dek mutants have been identified in maize and rice, and numerous genetic loci regulating grain fullness have also been discovered. Dek mutants, such as Dek10, Dek35, Dek36, Dek37, Dek39, Dek40, and Dek42 (Chen et al., 2017;Qi et al., 2017;Wang et al., 2017;Dai et al., 2018;Li et al., 2018;Ren et al., 2019;Zuo et al., 2019), display germinated mutant kernels that are lethal during the seedling stage; Dek15, Dek38, Dek41, and Dek44 (Lid et al., 2002;Garcia et al., 2017;He et al., 2019;Qi et al., 2019;Zhu et al., 2019) seeds cannot germinate at all, resulting in lethal embryo mutations. For example, the Dek15 gene encodes sister chromatid cohesion protein 4 (SCC4), and mutation of this gene disrupts the cell cycle and nuclear replication, leading to the complete failure of seed germination . ...
Wheat is a significant source of protein and starch worldwide. The defective kernel (Dek) mutant AK-3537, displaying a large hollow area in the endosperm and shrunken grain, was obtained through ethyl methane sulfonate (EMS) treatment of the wheat cultivar Aikang 58 (AK58). The mode of inheritance of the AK-3537 grain Dek phenotype was determined to be recessive with a specific statistical significance level. We used bulked segregant RNA-seq (BSR-seq), BSA-based exome capture sequencing (BSE-seq), and the ΔSNP-index algorithm to identify candidate regions for the grain Dek phenotype. Two major candidate regions, DCR1 (Dek candidate region 1) and DCR2, were identified on chromosome 7A between 279.98 and 287.93 Mb and 565.34 and 568.59 Mb, respectively. Based on transcriptome analysis and previous reports, we designed KASP genotyping assays based on SNP variations in the candidate regions and speculated that the candidate gene is TraesCS7A03G0625900 (HMGS-7A), which encodes a 3-hydroxy-3-methylglutaryl-CoA synthase. One SNP variation located at position 1,049 in the coding sequence (G>A) causes an amino acid change from Gly to Asp. The research suggests that functional changes in HMGS-7A may affect the expression of key enzyme genes involved in wheat starch syntheses, such as GBSSII and SSIIIa.
... For the luciferase complementation imaging (LCI) assays, the CDSs of ZmTFCB and its potential interactors were cloned into JW771 (NLUC) and JW772 (CLUC), respectively, by the In-Fusion reaction. The transformation and incubation processes were the same as previously described (He et al., 2019). The resulting luciferase signals were captured using the Tanon 5200 image system with a 10-min exposure. ...
Tubulin folding cofactors (TFCs) are required for tubulin folding, α/β tubulin heterodimer formation, and microtubule (MT) dynamics in yeast and mammals. However, the functions of their plant counterparts remain to be characterized.
We identified a natural maize crumpled kernel mutant, crk2, which exhibits reductions in endosperm cell number and size, as well as embryo/seedling lethality. Map‐based cloning and functional complementation confirmed that ZmTFCB is causal for the mutation.
ZmTFCB is targeted mainly to the cytosol. It facilitates α‐tubulin folding and heterodimer formation through sequential interactions with the cytosolic chaperonin‐containing TCP‐1 ε subunit ZmCCT5 and ZmTFCE, thus affecting the organization of both the spindle and phragmoplast MT array and the cortical MT polymerization and array formation, which consequently mediated cell division and cell growth. We detected a physical association between ZmTFCB and the maize MT plus‐end binding protein END‐BINDING1 (ZmEB1), indicating that ZmTFCB1 may modulate MT dynamics by sequestering ZmEB1.
Our data demonstrate that ZmTFCB is required for cell division and cell growth through modulating MT homeostasis, an evolutionarily conserved machinery with some species‐specific divergence.
... The maize gene Defective kernel 15 (Dek15) encodes a homolog of SISTER CHROMATID COHESION PROTEIN 4 (SCC4). The mitotic cell cycle and endoreduplication are disrupted in the dek15 mutant, resulting in reduced endosperm size and embryo lethality (He et al., 2019). ...
During grain filling, starch and other nutrients accumulate in the endosperm; this directly determines grain yield and grain quality in crops such as rice (Oryza sativa), maize (Zea mays), and wheat (Triticum aestivum). Grain filling is a complex trait affected by both intrinsic and environmental factors, making it difficult to explore the underlying genetics, molecular regulation, and the application of these genes for breeding. With the development of powerful genetic and molecular techniques, much has been learned about the genes and molecular networks related to grain filling over the past decades. In this review, we highlight the key factors affecting grain filling, including both biological and abiotic factors. We then summarize the key genes controlling grain filling and their roles in this event, including regulators of sugar translocation and starch biosynthesis, phytohormone‐related regulators, and other factors. Finally, we discuss how the current knowledge of valuable grain filling genes could be integrated with strategies for breeding cereal varieties with improved grain yield and quality.
... For example, the dek15 mutant exhibits a defective endosperm and embryo in the early stage, and the embryos appear to be more severely affected than the endosperm. The DEK15 encodes cohesin-loading complex subunit SCC4, and the loss of the SCC4 function leads to defects in the cell division and early kernel development [37]. Both embryo development and endosperm development are delayed in the smk4-1 mutant compared with that in the WT at 8 DAP. ...
The kernel serves as a storage organ for various nutrients and determines the yield and quality of maize. Understanding the mechanisms regulating kernel development is important for maize production. In this study, a small-kernel mutant smk7a of maize was characterized. Cytological observation suggested that the development of the endosperm and embryo was arrested in smk7a in the early development stage. Biochemical tests revealed that the starch, zein protein, and indole-3-acetic acid (IAA) contents were significantly lower in smk7a compared with wild-type (WT). Consistent with the defective development phenotype, transcriptome analysis of the kernels 12 and 20 days after pollination (DAP) revealed that the starch, zein, and auxin biosynthesis-related genes were dramatically downregulated in smk7a. Genetic mapping indicated that the mutant was controlled by a recessive gene located on chromosome 2. Our results suggest that disrupted nutrition accumulation and auxin synthesis cause the defective endosperm and embryo development of smk7a.
... Meiotic tassels of wild-type and transgenic plants were used for immunoblot analysis. Total proteins extraction and immunoblotting were performed as described previously (He et al., 2019). Antibodies to HA (BPI), GFP, plant ACTIN (ABclonal) and goat-anti-mouse immunoglobulin G antibodies conjugated to horseradish peroxidase (ABclonal) were diluted for 1 : 5000. ...
... Yeast two-hybrid (Y2H) and luciferase complementation imaging (LCI) assays were performed to determine the interactions of INVAN6 and INVAN6 Mei025 , and other proteins. The experiments were performed as previously described (He et al., 2019). The primers used here are listed in Dataset S1. ...
Faithful meiotic progression ensures the generation of viable gametes. Studies suggested the male meiosis of plants is sensitive to ambient temperature, but the underlying molecular mechanisms remain elusive.
Here, we characterized a maize (Zea mays ssp. mays L.) dominant male sterile mutant Mei025, in which the meiotic process of pollen mother cells (PMCs) was arrested after pachytene.
An Asp‐to‐Asn replacement at position 276 of INVERTASE ALKALINE NEUTRAL 6 (INVAN6), a cytosolic invertase (CIN) that predominantly exists in PMCs and specifically hydrolyses sucrose, was revealed to cause meiotic defects in Mei025. INVAN6 interacts with itself as well as with four other CINs and seven 14‐3‐3 proteins. Although INVAN6Mei025, the variant of INVAN6 found in Mei025, lacks hydrolytic activity entirely, its presence is deleterious to male meiosis, possibly in a dominant negative repression manner through interacting with its partner proteins. Notably, heat stress aggravated meiotic defects in invan6 null mutant. Further transcriptome data suggest INVAN6 has a fundamental role for sugar homeostasis and stress tolerance of male meiocytes.
In summary, this work uncovered the function of maize CIN in male meiosis and revealed the role of CIN‐mediated sugar metabolism and signalling in meiotic progression under heat stress.
... For kernel-related traits, many genes were characterized and cloned using comparative genomics and mutants, such as ZmGW2 (Li et al., 2010b), ZmGS3 (Li et al., 2010a), defective kernel (dek) mutants (Demko et al., 2014;Qi et al., 2017Qi et al., , 2019Wang et al., 2017;Dai et al., 2018;Li et al., 2018;He et al., 2019), MN2 (Guan et al., 2020), and ZmCEP1 . Many studies identified QTLs or associated SNPs to dissect the genetic mechanism of kernel-related traits. ...
Maize grain size is the main factor determining grain yield. Dissecting the genetic basis of maize grain size may help reveal the regulatory mechanism of maize seed development and yield formation. In this study, two associated populations were used for genome-wide association analysis of kernel length, kernel width, kernel thickness, and hundred-kernel weight from multiple locations in AM122 and AM180, respectively. Then, genome-wide association mapping was performed based on the maize 6H90K SNP chip. A total of 139 loci were identified as associated with the four traits with p < 1 × 10⁻⁴ using two models (FarmCPU and MLM). The transcriptome data showed that 15 of them were expressed differentially in two maize-inbred lines KB182 (small kernel) and KB020 (big kernel) during kernel development. These candidate genes were enriched in regulating peroxidase activity, oxidoreductase, and leaf senescence. The molecular function was major in binding and catalytic activity. This study provided important reference information for exploring maize kernel development mechanisms and applying molecular markers in high-yield breeding.
... Extraction of nuclei was carried out using a method described previously (Dolezel et al., 2007;He et al., 2019). Endosperm of five genotyped developing kernels of o18-1 and the wild type was pulled from the same segregated ears at 7, 10 and 15 DAP. ...
Riboflavin is the precursor of essential cofactors for diverse metabolic processes. Unlike animals, plants can de novo produce riboflavin through an ancestrally conserved pathway, like bacteria and fungi. However, the mechanism by which riboflavin regulates seed development is poorly understood. Here we report a novel maize (Zea mays L.) opaque mutant o18, which displays an increase in lysine accumulation but impaired endosperm filling and embryo development. O18 encodes a rate‐limiting bifunctional enzyme ZmRIBA1, targeted to plastid where to initiate riboflavin biosynthesis. Loss of function of O18 specifically disrupts respiratory complexes I and II, but also decreases SDH1 flavinylation, in turn shifts the mitochondrial tricarboxylic acid (TCA) cycle to glycolysis. The deprivation of cellular energy leads to cell cycle arrest at G1 and S phases in both mitosis and endoreduplication during endosperm development. The unexpected upregulation of cell cycle genes in o18 correlates with the increase of H3K4me3 levels, revealing a possible H3K4me‐mediated epigenetic backup mechanism for cell cycle progression under unfavorable circumstances. Overexpression of O18 increases riboflavin production and confers osmotic tolerance. Altogether, our results substantiate a key role of riboflavin in coordinating cellular energy and cell cycle to modulate maize endosperm development.
... The dek504-ref and WT kernels from a segregating F2 ear at 12, 15, and 18 DAP were prepared for cytological analysis according to previously published methods [60]. Paraffinembedded sections were stained with toluidine blue and observed under the Nikon Ti microscope (Nikon, Tokyo, Japan). ...
In flowering plants, RNA editing is a post-transcriptional process that selectively deaminates cytidines (C) to uridines (U) in organellar transcripts. Pentatricopeptide repeat (PPR) proteins have been identified as site-specific recognition factors for RNA editing. Here, we report the map-based cloning and molecular characterization of the defective kernel mutant dek504 in maize. Loss of Dek504 function leads to delayed embryogenesis and endosperm development, which produce small and collapsed kernels. Dek504 encodes an E+-type PPR protein targeted to the mitochondria, which is required for RNA editing of mitochondrial NADH dehydrogenase 3 at the nad3-317 and nad3-44 sites. Biochemical analysis of mitochondrial protein complexes revealed a significant reduction in the mitochondrial NADH dehydrogenase complex I activity, indicating that the alteration of the amino acid sequence at nad3-44 and nad3-317 through RNA editing is essential for NAD3 function. Moreover, the amino acids are highly conserved in monocots and eudicots, whereas the events of C-to-U editing are not conserved in flowering plants. Thus, our results indicate that Dek504 is essential for RNA editing of nad3, which is critical for NAD3 function, mitochondrial complex I stability, and seed development in maize.
