ZmZFP2 encoding a C4HC3-type RING zinc finger protein regulates kernel size and weight in maize

doi:10.21203/rs.3.rs-3718059/v1

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ZmZFP2 encoding a C4HC3-type RING zinc finger protein regulates kernel size and weight in maize

https://doi.org/10.21203/rs.3.rs-3718059/v1

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The development of maize (Zea mays) kernels is a complex physiological process, regulated by many genes in a spatio-temporal manner. However, there remain many unknown regulatory genes involved in maize kernel development. Here, we have identified a gene (ZmZFP2), which encodes a C4HC3-type RING zinc finger protein, that regulates maize kernel weight and size based on the suppression subtractive hybridization from maize endosperm in our previous studies. Through transgenic technology, over-expression of ZmZFP2 resulted in increased kernel weight and size compared to the wild type, while mutation of the gene led to decreased kernel weight and size. Microscopic observation of developing kernels revealed that ZmZFP2 affects the number of endosperm cells and their filling, as well as the starch and protein contents of the kernels. ZmZFP2 shows constitutive expression in various maize tissues. Furthermore, transcriptome analysis revealed that the zmzfp2-ems mutation significantly interfered with multiple biological processes, especially hormone pathways and ubiquitin degradation. Together, ZmZFP2 affects kernel size and weight by influencing endosperm cell number and filling, providing a valuable potential gene for increasing maize yield.

maize

RING zinc finger

endosperm development

filling

kernel size and weight

ZmZFP2 plays a positive role in the regulation of kernel weight and size, and is implicated in the control of starch and protein accumulation in maize kernels.

Maize is one of the most widely cultivated and high-yielding crops in the world, serving as a staple food, animal feed, and industrial raw materials (Liu, et al. 2020a). The kernel of maize is a storage organ consisting of essential components for plant growth and reproduction. Maize kernel is composed of the diploid embryo and the triploid endosperm that develop from double fertilization and are enclosed within the maternally derived pericarp (Fu, et al. 2023; Walley, et al. 2013). Kernel size affects not only evolutionary fitness but also grain yield of crops (Li, et al. 2019). Kernel weight contains about starch, protein, lipid, and micronutrients such as vitamins and minerals (Dai, et al. 2021; Nuss and Tanumihardjo 2010).Several signaling pathways have been shown to control kernel size and weight by regulating the growth of maternal tissues. These include or involve the ubiquitin-proteasome pathway, G-protein signaling, mitogen-activated protein kinase (MAPK) signaling, phytohormone perception and homeostasis, and some transcriptional regulators (Li, et al. 2019).

The ubiquitin-proteasome pathway is one of the important regulating pathways of kernel size and weight in different cereal crops (Long, et al. 2023). The ubiquitination of protein substrate involves three distinct enzymes, ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin protein ligase (E3) (Moon, et al. 2004). Substrate specificity is largely determined by the E3 ubiquitin ligases, which mediates the recruitment of proper substrates. Ubiquitin-dependent protein modification has been shown to play important roles in many cellular processes, such as hormone regulation, growth, development, senescence and stress responses (Li and Li 2014; Ling, et al. 2019; Varshney and Majee 2022). Several studies have reported that most RING finger proteins are E3 ubiquitin ligases in vitro, which target proteins for ubiquitin-dependent degradation by the 26S proteasome and play important roles in diverse aspects of cellular regulation in plants (Disch, et al. 2006; Park, et al. 2010; Saxena, et al. 2023; Wang, et al. 2020b; Wu, et al. 2014; Xia, et al. 2012).

RING Zinc finger proteins are widely distributed in eukaryotes. They possess a characteristic RING zinc finger domain, forming a finger-like spatial conformation by binding to zinc ions (Han, et al. 2020). There are 477 and 616 and RING zinc finger domain genes in Arabidopsis and human respectively (Deshaies and Joazeiro 2009; Vierstra 2009). These proteins participate in various physiological activities, including growth, development, signaling transduction, and environmental adaptation (Gasparini, et al. 2023; Joseph, et al. 2014; Liu, et al. 2022; Liu, et al. 2020b). The RING finger domain of RING zinc finger proteins serves as a site for protein-protein interaction and is essential for their E3 ligase activity. The conserved sequence of RING zinc finger proteins is defined as Cys-X₂-Cys-X₍₉₋₃₉₎-Cys-X₍₁₋₃₎-His-X₍₂₋₃₎-Cys/His-X₂-Cys-X₍₄₋₄₈₎-Cys-X₂-Cys, where X represents any amin acid, and the number of X residues varies among different fingers (Borden and Freemont 1996). RING zinc finger proteins can be mainly categorized into 13 types: RING-H2, RING-HC (RING-HCa and RING-HCb), RING-v, RING-C2, RING-D, RING-S/T, RING-G, RING-mh2, RING-mhc, C2SHC4, C3GC3S, C2HC5, and C3HCHC2 (Sun et al., 2019). Among them, RING-HC (C3HC4) and RING-H2 (C3H2C3 and C4HC3) types are the most abundant (Stone, et al. 2005).

Several studies have reported the functions and mechanisms of RING zinc finger proteins. The loss of function of the RING zinc finger protein GW2 gene in rice results in increased cell number, leading to a larger (wider) spikelet hull, accelerated grain milk filling rate, and increased grain width, weight, and yield. OsGW2 regulates cell division by targeting substrates to the proteasome for degradation, thereby negatively regulating (Song, et al. 2007). In wheat, the TaGW2 gene regulates grain size by controlling endosperm cell number (Bednarek, et al. 2012). The RING zinc finger protein E3 ubiquitin ligase BIG BROTHER acts as a negative regulator of plant organ growth, mediating degradation of key growth stimulatory factors (Disch, et al. 2006). Inhibition of RING zinc finger protein DAF expression leads to altered pollen development, causing anthers to fail to dehisce and resulting in female sterility in Arabidopsis (Peng, et al. 2013). OsDSG1 regulates rice grain germination and stress response (Park, et al. 2010). PEX10 (Peroxin 10) encodes a peroxisomal membrane protein involved in photorespiration and peroxisome attachment to chloroplasts (Schumann, et al. 2007). RIE1 (RING zinc-finger protein gene for embryogenesis 1) plays a critical role in seed development, and its loss of function causes embryo lethality from the globular to late torpedo stage in Arabidopsis (Xu and Li 2003). However, there are few reports on the functional analysis of maize zinc finger proteins. ZmRFP1 is involved in ABA-dependent drought stress response (Xia, et al. 2012), while ZmXERICO enhances drought tolerance in transgenic tobacco by improving water retention capacity, reducing ROS accumulation, and enhancing the antioxidant system to alleviate membrane damage (Gao, et al. 2012). Maize possesses homologs of two OsGW2 genes, ZmGW2-CHR4 and ZmGW2-CHR5, which play a role in grain size and weight variation (Li, et al. 2010).

In our previous studies, a candidate EST (PE12C5) was isolated from four suppression subtractive hybridization (SSH) libraries constructed from the key developmental stages of endosperm in two maize inbred lines, a regular dent inbred Dan232 (large-sized kernel) and a popcorn inbred N04 (small-sized kernel) (Li, et al. 2012; Liu, et al. 2010). In this study, we identified ZmZFP2 as a regulatory factor for kernel development. Sequence analysis showed that ZmZFP2 protein comprises a highly conserved zinc finger domain. Over-expression of ZmZFP2 in maize significantly increased kernel weight and width. Histological analysis revealed that ZmZFP2 plays a role in the regulation of kernel development through cell division and cell size. Our results indicate that ZmZFP2 controls kernel weight and size by affecting endosperm cell number and filling process.

Identification zmzfp2 mutant form small kernels

In our previous study, we isolated a candidate gene, RING Zinc Finger Protein (ZmZFP2, Zm00001d033780), from two crucial developmental stages of endosperm in a dent maize inbred Dan232 with large-sized kernels, and a popcorn inbred N04 with small-sized kernels (Li, et al. 2012; Liu, et al. 2010). ZmZFP2 comprises six exons and a C4HC3-type RING zinc finger domain (Fig. S1A-B). We identified a mutant for ZmZFP2 alleles in the Maize EMS induced Mutant Database (MEMD) (http://www.elabcaas.cn/memd/) (Lu, et al. 2018). The zmzfp2-ems allele is caused by a typical EMS-induced C to G mutation, resulting in a codon change of CAG (Arginine) to TAG (stop codon) within the open reading frame (Fig. S1A and C), leading to premature termination of the mutant allele translation (Fig. S1B). Subsequently, homozygous zmzfp2-ems mutant and corresponding wild-type (WT) were selected from self-crossed heterozygous mutants. The zmzfp2-ems mutant plants exhibited a significant decrease in plant height compared to the WT (Fig. 1A and 1F), while the phenotypic difference in ear appearance was not apparent. Quantitative analysis revealed that the 100-kernel weight of zmzfp2-ems decreased by 34.9%, and the length and width of the kernels were reduced by 9.6% and 17.0% respectively, compared to the WT (Fig. 1B-D and 1G-I). Quantitative RT-PCR analysis showed that the transcript levels of ZmZFP2 in the zmzfp2-ems mutant were lower than in its corresponding WT (Fig. 1E). These results indicate that the EMS-induced zmzfp2-ems mutant affects kernel weight and size.

The function of ZmZFP2 in maize transgenic lines

We employed the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9) technology strategy to target two sites in the second exon of ZmZFP2 and performed genetic transformation in the maize inbred line B104 (Fig. 2A-B). Two CRISPR editing events, zmzfp2-cr1 and zmzfp2-cr2, were generated, with zmzfp2-cr1 showing a 1-bp insertion within the open reading frame and zmzfp2-cr2 showing a 2-bp deletion (Fig. 2A). Phenotypic analysis of the kernels revealed that both zmzfp2-cr1 and zmzfp2-cr2 exhibited significant reductions in kernel 100-kernel weight, length and width compared to their respective WT controls, with reductions of 26.0% and 21.6% in 100-kernel weight, 17.4% and 7.7% in length, and 17.2% and 12.9% in width, respectively (Fig. 2C-D and E-G). These phenotypes were similar to those observed in the zmzfp2-ems mutant.

In Arabidopsis, transgenic plants over-expressing ZmZFP2 under the control of the CaMV35S promoter exhibited a significant increase in leaf area and plant height compared to WT plants (Fig. S2A-B and D-E). Additionally, the 1000-seed weight, seed width, and seed length of Arabidopsis were significantly enhanced (Fig. S2C and F-G). Next, we over-expressed ZmZFP2 in the maize inbred line B104 under the control of a maize ubiquitin promoter (Ubi::ZmZFP2). We assessed the agronomic traits of transgenic over-expression ZmZFP2 plants in the T₂ generation, and the ear morphology was slightly larger than that of the WT (Fig. 3A). The transcription levels of ZmZFP2 in the over-expressing cell lines were 3-fold, 7-fold, and 2-fold higher than the WT (Fig. 3D). In comparison to the WT, over-expression of ZmZFP2 in maize increased the 100-kernel weight and kernel length, with little change in the kernel width (Fig. 3B-C and E-G). Statistical analysis showed that the 100-kernel weight in over-expression lines, ZmZFP2-OE1, OE2, and OE3, was enhanced by 5.9%, 9.8%, and 6.6%, respectively (Fig. 3E). Agronomic traits of the three OE lines collectively indicate that over-expressing ZmZFP2 significantly increases kernel weight and enlarges kernel size. These results, in combination with the results in the zmzfp2 mutants, suggest that ZmZFP2 positively regulates kernel weight and size.

The effect of ZmZFP2 on endosperm cell phenotype, starch, and protein accumulation

Given that ZmZFP2 affects the size and weight of maize kernels, the development process of the kernels is determined by the division and expansion of endosperm cells (Dante, et al. 2014; Ji, et al. 2022). We analyzed the longitudinal sections of the endosperm of kernels at 6, 8 and 16 DAP (days after pollination) (Fig. 4A-B and 4G-H). The morphology of the endosperm of the zmzfp2-ems mutant (Fig. 4A-B), zmzfp2-cr1 mutant (Fig. 4G), and ZmZFP2-OE2 (Fig. 4H) showed significant differences compared to their corresponding WT, with Fig. 4A and Fig. 4B being the magnified views of Fig. 4D and Fig. 4E, respectively. From the figures, it can be observed that the grain-filling of the endosperm in the zmzfp2-ems and zmzfp2-cr1 mutants were lower than that of the WT, particularly at the top region, in which the difference between the kernels at 8 DAP and their corresponding WT was more significant compared to that of the 16 DAP kernels and their corresponding WT. We found that the size of the endosperm in the zmzfp2-ems mutant and zmzfp2-cr1 mutant were smaller than that of the WT at both 6, 8 and 16 DAP. Statistical analysis revealed a significant reduction in the number of endosperm cells in the zmzfp2-ems mutant and zmzfp2-cr1 mutant compared to the WT, while the cell size difference was not significant (Fig. 4C-F and 4I-J).

By observing the top region of the endosperm at 16 DAP through transmission electron microscopy, the starch granules of the zmzfp2-ems mutant were smaller and more irregular compared to the WT (Fig. 5A), and the protein bodies were also smaller (Fig. 5B). In the mature dry kernels of the zmzfp2-ems mutant and zmzfp2-cr1 mutants, the starch contents were reduced by 5.1% and 6.6% respectively compared to the WT, and the total protein content decreased by 8.9% and 6.2% respectively (Fig. 5C-D and 5E-F). The content of zein in the zmzfp2-ems and zmzfp2-cr1 mutants significantly decreased, while the difference in non-zein content was not significant (Fig. S3A-D). The starch, total protein, and zein content in the mature kernels of ZmZFP2-OE2 increased by 4.8% and 7.6% respectively. These results indicate that the decrease in kernel size and weight caused by ZmZFP2 may be affected by the number of endosperm cells, as well as related to the accumulation of starch and protein.

Expression profile and subcellular localization of ZmZFP2

We employed quantitative RT-PCR analysis to measure the expression levels of ZmZFP2 in the inbred line B73, allowing to analyze the spatio-temporal expression patterns of ZmZFP2 in different tissues and developmental stages of maize. The highest expression of ZmZFP2 was observed in the shoot apical meristem (SAM) and the ear, with detectable expression in kernels at different developmental stages (Fig. 6A). Additionally, we utilized RNA in situ hybridization to explore the expression of ZmZFP2 in kernels at 10 and 15 DAP. Figure 6B showed abundant expressed of ZmZFP2 in the apical region of the embryo in the kernel, with the highest expression observed in the 15 DAP embryo (Fig. 6B). These in situ hybridization results were consistent with the developmental morphology of ZmZFP2 in the endosperm during grain-filling (Fig. 6A, Fig. 4A-B).

We conducted a search of the National Center for Biotechnology Information (NCBI) database and identified orthologs of ZmZFP2 in eudicots including soybean (Glycine max) and cotton (Gossypium hirsutum), and monocots including Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), millet (Setaria italica) and sorghum (Sorghum bicolor) (Fig. S4). The multiple sequence alignment results showed high similarities between the ZmZFP2 protein and its orthologs, with amino acid sequence similarities of 70%, 88%, 94%, 94% and 84% with sorghum and Arabidopsis, rice, millet, sorghum and wheat, respectively (Fig. S5A). Additionally, an analysis of the conserved domain demonstrated that the ZmZFP2 protein contained a single RING domain at the N-terminal and three putative transmembrane domains in its C-terminal region, which were conserved (Fig. S5A). The consensus sequence of RING zinc finger proteins Cys-X₂-Cys-X₍₉₋₃₉₎-Cys-X₁-Cys/His-X₂-Cys-X₍₄₋₄₈₎-Cys-X₂-Cys was highly conserved across different species (Fig. S5A).

To investigate the subcellular localization of ZmZFP2, the full-length ZmZFP2 protein were fused to the N terminus of green fluorescent protein (GFP). The resulting constructs driven by the constitutive 35S promoter were first transiently expressed in tobacco (Nicotiana benthamiana) leaves. ZmZFP2-GFP signals appears to be closely around the DAPI (4',6-diamidino-2-phenylindole) in the nucleus (Fig. 6C). To further determine ZmZFP2 localization, similar around the nucleus localization of ZmZFP2-GFP was observed in maize leaf mesophyll protoplasts (Fig. 6D). Using TMHMM 2.0 analysis (https://services.healthtech.dtu.dk/services/TMHMM-2.0/), we identified three transmembrane domains in ZmZFP2 (Fig. S5A). It revealed that ZmZFP2-GFP containing the entire ZmZFP2 protein localizes to the nuclear outer membrane. Using the yeast GAL4 system, the result showed ZmZFP2 has no intrinsic activation properties (Fig. S5B). Signal peptide was not predicted in ZmZFP2 protein sequence using SignalP 3.0 (https://services.healthtech.dtu.dk/services/SignalP-3.0/). These results showed that ZmZFP2-GFP distinctly localized to the nuclear outer membrane without transcriptional activation.

Transcriptome Analysis of Genes Regulated by ZmZFP2

To access the changes in gene expression associated with ZmZFP2, we performed a transcriptome analysis using RNA-seq on 8-DAP kernels of zmzfp2-ems and WT. A total of 266 upregulated and 1156 downregulated transcripts were identified in the zmzfp2-ems relative to the WT, indicating that ZmZFP2 has a more positive effect on gene expression than a negative effect (Fig. 7A). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of all the differentially expressed genes revealed that enrichment of genes was mainly involved in organic metabolism, compound biosynthesis, and signaling pathways (Fig. 7B; Supplemental Data 1). For instance, four genes involved in the MAPK signaling pathway in plant were also strongly enriched in plant hormone signaling transduction (Fig. 7C; Supplementary Data 2). In addition, four genes, including Zm00001d005823 (flavonoid 3'-monooxygenase), Zm00001d020628 (trimethyltridecatetraene synthase-like), Zm00001d022351, Zm00001d053617 (brassinosteroid catabolism 1) were significantly downregulated and enriched in brassinosteroid biosynthesis (Fig. 7B-C). Zeatin is a common plant cytokinin and plays a crucial role in cell division. We found that the differentially expressed genes were enriched in zeatin biosynthesis (Fig. 7B-C), including Zm00001d000237 (cis-zeatin O-glucosyltransferase 1), Zm00001d012335 (cis-zeatin O-glucosyltransferase 2), Zm00001d012641 (cytokinin oxidase4b), Zm00001d016417 (UDP-glycosyltransferase), Zm00001d032664 (cytokinin oxidase6), and Zm00001d039643 (cytokinin-O-glucosyltransferase 3), indicating that ZmZFP2 regulates kernel width via cell division. Starch and sucrose metabolism were also significantly enriched, including genes Zm00001d014083 (beta amylase), Zm00001d018082 (trehalose-phosphate phosphatase 4), Zm00001d025354 (invertase cell wall5), Zm00001d032118 (trehalose-6-phosphate synthase3), and Zm00001d035037 (fructokinase2). All of these genes were found to be downregulated in zmzfp2-cr1 compared with WT (Fig. S6A-D). Endoplasmic reticulum associated degradation (ERAD) is a primary method to degrade proteins to maintain cell homeostasis, which requires the ubiquitination of substrate proteins by E1 (ubiquitin-activating enzyme), E2s (ubiquitin-conjugating enzymes) and E3s (ubiquitin E3 ligases) (Chen, et al. 2020). We found that eleven differentially expressed genes were significantly enriched in protein processing in the endoplasmic reticulum, which is consistent with its ubiquitin ligase function (Fig. 7B).

Improving maize yield is a primary objective for breeders. In this study, we identified a C4HC3-type zinc finger E3 ubiquitin ligase gene, ZmZFP2, which regulates kernel weight. ZmZFP2 encodes a conserved E3 ubiquitin ligase that positively regulates kernel weight and width. Our findings elucidate the role of ZmZFP2 in endosperm development and its impact on starch and protein accumulation in kernels. While the ubiquitin-proteasome pathway has been reported to play a crucial role in grain shape regulation(Li and Li 2014; Li, et al. 2019; Long, et al. 2023), the functions of ZmZFP2 and its homologous genes in kernel weight and shape have not been identified and characterized until now. Here, we report that ZmZFP2 influences kernel weight and width, suggesting that the ubiquitination pathway may regulate kernel weight and width through novel mechanisms.

The cell division and proliferation of maize kernels primarily occur during early development, starting at approximately 12 DAP, when starch and storage proteins begin to accumulate along with cell expansion (Sabelli and Larkins 2009). ZmZFP2 is expressed at different stages of maize kernel development, from 1 DAP to 30 DAP, and exhibits constitutive expression in various tissues and organs (Fig. 6A). This suggests that ZmZFP2 may play a crucial role throughout the process of maize kernel development. Most zinc finger proteins are E3 ubiquitin ligases involved in ubiquitin-dependent degradation mediated by the 26S proteasome (Deshaies and Joazeiro 2009; Jackson, et al. 2000; Li and Li 2014). These E3 ligases not only participate in the regulation of grain development in plants but also play important physiological roles in various cellular regulations (Franciosini, et al. 2013; Li, et al. 2023; Saxena, et al. 2023; Wang, et al. 2020b).

The content of zein is reduced in the zmzfp2-ems mutant, while the content of non-zein remains relatively constant (Fig. S3). ZmZFP2 may regulate gene expression or protein translation related to zein synthesis during maize kernel development, leading to the inhibition of zein synthesis in the zmzfp2-ems mutant and resulting in a lower zein content. In the WT, the normal function of ZmZFP2 may contribute to zein synthesis and folding, forming stable packaging structures within the protein bodies. In the zmzfp2-ems mutant, the abnormal function ZmZFP2 may prevent zein from properly folding into stable structures, thereby affecting its accumulation levels in the kernel. Mutations in zmzfp2-ems may cause changes in protein structure or stability, which can affect the structure or stability of certain proteins, resulting in changes in their zein accumulation levels in the kernel. For example, the content of α19 and α22 zein is lower in the zmzfp2-ems mutant compared to the WT (Fig. S3A). The content of non-zein proteins shows little difference between the WT and the zmzfp2-ems mutant, suggesting that these metabolic pathways may not be significantly affected. This indicates that ZmZFP2 primarily affects pathways related to zein synthesis, while having a relatively smaller effect on non-zein protein synthesis. Zein is the main storage protein in maize kernels, and the size and weight of maize kernels are typically closely related to zein content (Li and Song 2020; Wang, et al. 2020a). Therefore, the decrease in zein in the zmzfp2-ems mutant may result in smaller kernel size and reduced kernel weight. Zein is one of the main nutritional reserves in maize kernels, providing energy and nitrogen sources during kernel development. If the zmzfp2-ems mutation leads to reduced zein synthesis and accumulation, it indicates that the nutrient reserves within the kernels may be affected, resulting in smaller kernel size and reduced kernel weight. In addition to changes in zein, the zmzfp2-ems mutant may also have an impact on other metabolic pathways and developmental processes. These effects may be direct, such as regulating the synthesis or transportation of other key proteins during kernel development, or indirect, such as affecting the biosynthesis pathways of kernel lipids and starch. The combined effect of these factors may contribute to the decrease in kernel size and weight.

ZmZFP2 is predicted to harbor a C4HC3-type RING zinc finger domain and three transmembrane domains (Fig. 6B; Fig. S5A). The C4HC3-type zinc finger domain typically comprises a protein structure composed of four cysteine (C4) and three histidine (H3) residues (Deshaies and Joazeiro 2009; Stone, et al. 2005). This domain primarily functions to coordinate with the metal zinc ions, forming a stable RING-like structure that allows the protein to interact with other proteins or DNA sequences and participate in various biological processes, including gene regulation, transcription, transcription activation, DNA repair, etc (Ban and Estelle 2021; Zheng and Shabek 2017). The zinc finger domain recognizes and binds specific DNA sequences through its unique amino acid composition and spatial structure, regulating the expression of target genes (Chai, et al. 2022; Kong, et al. 2021; Yin, et al. 2020). Moreover, it can also regulate signal pathways, cell apoptosis, cell cycle, and other biological processes by interacting with other proteins (Cui, et al. 2018; Zhou, et al. 2013). Therefore, we hypothesized that ZmZFP2 may be subcellularly localized in the nuclear outer membrane, while also possibly being localized in the cell membrane due to the predicted transmembrane domains. However, experiments exploring the subcellular localization of ZmZFP2 were inconclusive, and thus, the experimental results could not be presented in this paper. This is an intriguing topic that necessitates the optimization of our experimental concepts and methods. We propose that if ZmZFP2 is localized in the cell membrane, the hydrophobic nature of the membrane might constrain the direct interaction between the protein and DNA. The binding function of the C4HC3 domain in on the cell membrane might be limited, but this does not imply that it entirely loses its DNA-binding capacity. In such a case, other protein parts might be involved to establish the connection between the protein and DNA. For instance, other domains could interact with DNA or other mediator factors to mediate the interaction between the C4HC3 domain and DNA (Cao, et al. 2016; Cheung, et al. 2007). This interaction might require the participation of other cellular components to ensure efficient binding of the C4HC3 domain to DNA. Furthermore, if ZmZFP2 is subcellularly localized in the nuclear outer membrane, the predicted transmembrane domain could potentially cause the protein to have multiple subcellular localizations within the cell, with nucleolar enrichment under specific experimental conditions. There is also a possibility that the transmembrane domain is non-functional or triggered by other protein modifications. In this scenario, the presence of the transmembrane domain might be retained due to genomic evolution, but its function remains unclear in the current context. Additionally, under certain physiological conditions, the protein can alter its subcellular localization through interactions with other proteins or specific factors.

It is evident that the ZmZFP2 gene holds promise for enhancing maize yield. In conclusion, our research results suggest that ZmZFP2 plays a positive role in the regulation of kernel weight and size, and is implicated in the control of starch and protein accumulation in kernels. This positions ZmZFP2 as a prospective candidate gene for future endeavors aimed at boosting maize yield.

Plant materials and growth conditions

An EST (PE12C5) putatively encoding ZmZFP2 was isolated from the suppression subtractive hybridization (SSH) library constructed between 10 and 20 DAP endosperm in a dent maize inbred Dan232 with large-sized kernels, and a popcorn inbred N04 with small-sized kernels (Liu, et al. 2010). The zmzfp2 mutant (accession number EMS3-013346) was obtained from the Maize EMS-induced Mutant Database (MEMD) (http://www.elabcaas.cn/memd/). Immature kernels were harvested at time points of 1, 3, 6, 8, 10, 12, 14, 16, 18, 20, 24, and 30 DAP for expression pattern analysis, RNA in situ hybridization, and RNA-seq assays. Manual dissection was used to collect embryo, endosperm, whole seed, root, stem, leaf, and SAM sample from at least three plants. These samples were frozen in liquid nitrogen and stored at -80°C.

All maize plants were grown in the field in the Chinese cities of Zhengzhou (34.9° N, 113.6° E), Shanghai (30.5° N, 121.1° E), or Sanya (18.2° N, 109.3° E). To minimize the effect of environmental factors, we phenotyped and genotyped kernel weight and size at these three sites. Kernels selected from the middle region of ears uniform kernel set were used for photography and kernel trait measurement, and at least 100 kernels were measured for each ear. The kernel weight, length, and width were measured using an image analysis method provided with SC-E software (Wanshen Detection Technology, Hangzhou, China). The kernel thickness was measured using an electronic digital display vernier caliper with fully filled kernels.

Seeds of Arabidopsis were sterilized with a 6.25% sodium hypochlorite solution, and then germinated on Murashige and Skoog basal medium (MS, Sigma-Aldrich) supplemented with 0.8% (w/v) agar and 3% sucrose. Seven-day-old seedlings were transplanted into potting soil within a chamber (photosynthetic photon flux density of 150 µmol of photons m^− 2s^− 1, 16-h-light/8-h-dark cycle at 22°C).

Plasmid construction and plant transformation

We obtained overexpression and knockout transgenic maize mutants via Agrobacterium tumefaciens-mediated transformation by WiMi Biotechnology (http://www.wimibio.com/) in Jiangsu, China. For the overexpression lines, we amplified the full-length of ZmZFP2 and inserted it into a vector driven by a ubiquitin promoter (Tian, et al. 2019). The construct was subsequently introduced into A. tumefaciens EHA105 and transformed into B104. Following the standard transformation protocol, three positive events were recovered. For the knockout mutants, we searched for single guide RNA (sgRNA) sites in ZmZFP2 using the website (http://cbi.hzau.edu.cn/CRISPR2/) and selected the highest On-score. A sgRNA targeting exon 2 (+ 260 to + 279) was cloned into the PsiI and XbaI sites between the maize U6 promoter and U6 terminator (Ji, et al. 2022). The constructs were introduced into A. tumefaciens EHA105 and transformed into B104. To identify Cas9-free transgenic plants with homozygous mutations, we utilized PCR products sequencing and hygromycin selection. The primers used for constructions and genotyping are listed in Tables S1.

For the over-expression of ZmZFP2 in Arabidopsis transformation, the ZmZFP2 coding sequence was amplified with specific primers and cloned into the pCAMBIA3301 plant expression vector, driven by the CaMV 35S promoter. Transformed maize immature embryos and Arabidopsis seeding were screened using basta (7 mg/ml), followed by further validated via PCR using forward primer OE-ZFP2-F and reverse primer OE-ZFP2-R (Table S1). The selected plant individuals were used for self-pollination, a total of eight independent events. All primers used in the study are listed in Table S1.

RNA extraction and quantitative real-time PCR analysis

Total RNA was extracted from root, stem, leaf, shoot apical meristem (SAM), tassel, endosperm, and seed using Trizol reagent (Invitrogen, Los Angeles, USA). Fresh tissue was collected and suspended in 0.3 mL of RNA extraction buffer (150 mM LiCl, 50 mM Tris pH 8.0, 5 mM EDTA pH 8.0, and 1% SDS [w/v]) in a 2 mL RNase-free tube. The homogenized samples were extracted twice using 0.3 mL phenol-chloroform (pH 4.2) and once with 0.3 mL chloroform. Subsequently, RNA was extracted with 1 mL Trizol regent (Invitrogen, Los Angeles, USA). The extracted total RNA was further purified using the RNeasy Mini Kit with DNaseI digestion (Qiagen, Dusseldorf, Germany). An equal amount of RNA was selected for reverse transcription in each sample with the ImProm-IITM Reverse Transcription System (Promega, Madison, USA). Quantitative RT-PCR was performed with SYBR Green I (Yeasen, Shanghai, China). The relative expression levels were calculated using the 2^−ΔΔCT method.

Histocytochemical Analysis

The material used for in situ hybridization was embedded in paraffin. Fresh tissue was taken and fixed in 4% paraformaldehyde solution with 0.1% Triton X-100 and 0.1% Tween 20 in PBS (Sangon Biotech, Shanghai, China). Kernel slices of 10 mm thickness were placed on an adhesive slide. Semi-thin sections embedded in resin were used for SEM. Kernels were fixed in FAA buffer (50% ethanol: formaldehyde: acetic acid = 90:5:5 [v:v:v]) and embedded in resin after ethanol gradient dehydration. The 2 mm sections were stained with toluidine blue solution and then photographed under a bright field using a Leica DM2500 (Leica, Heidelberg, Germany). Ultrathin sections were used to observe changes in amyloid and protein bodies. The tissue was cut from about 5 layers of tissue inside the aleurone layer and fixed in 2.5% glutaraldehyde in phosphate buffer pH 7.2. The 1 mm ultrathin sections were imaged at 80 KV with a Hitachi H-7650 transmission electron microscope (Leica, Heidelberg, Germany).

RNA in Situ Hybridization

In situ hybridization was conducted using 10 and 15 DAP kernels of B73. A ZmKW1 fragment was amplified by PCR and then inserted into the pSPT18 vector. The sense and antisense RNA probes were produced in vitro using T7 and SP6 RNA polymerase with DIG RNA Labeling Mixture (Roche, Basel, Switzerland). Tissue processing and in situ hybridization experiments using 10-mm sections were performed according to the methods described previously (Zhang, et al. 2015). The sections were observed and imaged using a light stereomicroscope (M165 FC, Leica, Heidelberg, Germany).

Measurement of the proteins and starch

The total protein content was determined by Rapid N EXCEED instrument (elementar, Frankfurt, Germany) (Huang, et al. 2022). mature kernels, with the pericarp and embryo removed, were dried at 42°C for at least 12 hours, and then ground to a fine powder using a tissue grinder. Approximately 50mg of the powder was weighed and wrapped in specialized tinfoil for elemental analysis. The protein content was then calculated from nitrogen content using Kjeldahl method. For each sample, 100mg of powder was used to determine the content of zein and non-zein.

The extraction and analysis of zein and non-zein proteins was conducted as described previously (Yang, et al. 2018). The total protein content was determined using the Total Starch Assay Kit (K-TSTA; Megazyme, Bray, Ireland) according to the manufacturer’s procedure. All measurements were replicated at least three times.

Sequence alignment and phylogenetic analysis

Orthologs of the ZmZFP2 protein in various species were identified in the NCBI database through a BLASTp search. Multiple sequences were aligned using ClustalX 2.1 with default parameters. For the phylogenetic analysis, amino acid sequences were aligned with ClustalW, and the phylogenetic trees were constructed using the MEGA7.0 software package with the bootstrap value of 1000 replicates.

Subcellular localization

To investigate the subcellular localization of ZmZFP2. The amplified ZmZFP2 coding sequence was inserted into a pCAMBIA1301 plasmid driven by the 35S promoter. The constructed vector was transformed into Agrobacterium tumefaciens GV3101 (Weidibio, Shanghai, China) and injected into tobacco leaves. The transfected leaves were observed using an LSM880 confocal microscope under Airyscan modes (Zeiss, Jena, Germany). We also verified ZmZFP2 localization in maize leaf protoplast system. Over 1ug/ul the constructed vector was extracted with NucleoBond Xtra Midi (MN, Munich, Germany) according to the manufacturer’s instructions. Protoplasts were obtained from maize leaves cultured in dark for about 3 weeks.

RNA-seq analysis

RNA-seq libraries from three biological replicates kernel at 8-DAP in WT and zmzfp2 were prepared with an Illumina Standard library preparation kit and sequenced by HiSeqTM 2500 at Novogene Biotech Co, Ltd (Novogene Biotech,Tianjin, China). The six DNA-free RNA samples were sequenced, yielding 20 Gb of raw sequencing data. Clean reads were aligned to the B73 reference genome (RefGen_v4) and the reference gene model dataset (FGS 5b) using TopHat/Bowtie2N (ccb.jhu.edu/software/tophat/). The gene expression value was calculated as fragments per kilobase of transcript per million mapped reads (FPKM). The FPKM value should be greater than zero for it to be considered as expressed gene in the three biological replicates. Finally, differentially expressed genes between the WT were identified based on their corrected P-values.

Statistics and reproducibility

Association analysis was performed with Fisher’s exact test, which is powerful in analyzing qualitative traits such as kernel weight. The data are presented as mean ± standard deviation (s.d.). Statistical analyses were carried out using GraphPad 8.0.2 and Microsoft Excel 2019 software. The significance of differences among different groups was assessed by one-way ANOVA with a Tukey’s test and two-sided Student’s t-test. A P-value of less than 0.05 was considered statistically significant. Detailed statistical information can be found in the Figure legends.

Author contribution statement

YL, YW and YD conceived and supervised the study. YL, LZ and QW designed and performed the experiments. LZ and QZ performed RNA sequence analysis, LZ and MF performed RNA in situ hybridization and analyzed the gene expression. LZ, QW, MF, HW, XL, YW, LH and WY constructed the CRISPR/Cas9 and overexpression experiments and performed ZmZFP2 function analysis. LZ, QW, WL, QZ, YD, YW and YL analyzed the data, prepared the figures, wrote and revised the manuscript. All authors read and approved the manuscript.

Acknowledgements

We thank Chen Ji, Jiechen Wang, Qiong Wang, Yongcai Huang, and Haihai Wang (Center for Excellence in Molecular Plant Sciences, CAS) for their support in maize cultivation and technical assistance. We thank all members in our laboratory for critical reading and discussion.

Funding

This work was supported by grants from the National Natural Science Foundation of China (31971962, 31771812 and 32272129 to YL), Zhongyuan Scholars in Henan Province (22400510003 to YL), the Major Public Welfare Projects of Henan Province (201300111100 to YL), Tackle Program of Agricultural Seed in Henan Province (2022010201 to YL), Technical System of Maize Industry in Henan Province (HARS-22-02-S to YL), and Key Scientific Research Projects for Higher Education of Henan Province (19zx001 to YL).

Data availability

Data supporting the findings of this work are available within the paper and its Supplementary Information files. A reporting summary for this Article is available as a Supplementary Information file. The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request. RNA-seq data was deposited in in the National Center for Biotechnology Information (NCBI) under the SRA accession number PRJNA970054.

Supplemental information

Supplemental information is available at Plant Cell Reports Online.

Conflict of interest

The authors declare no conflicts of interest.

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SupplementaryDataset1.xls
Supplementary Dataset 1 KEGG enrichment analysis of differentially expressed genes between WT and zmzfp2-ems.
SupplementaryDataset2.xlsx
Supplementary Dataset 2 Differentially expressed genes in enrichment of WT and zmzfp2-ems.
ZmZFP2SupplementaryFigures.pdf

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ZmZFP2 encoding a C4HC3-type RING zinc finger protein regulates kernel size and weight in maize

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Abstract

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Key Message

Introduction

Results

Expression profile and subcellular localization of ZmZFP2

Discussion

Materials and methods

Plant materials and growth conditions

Plasmid construction and plant transformation

RNA extraction and quantitative real-time PCR analysis

Histocytochemical Analysis

Measurement of the proteins and starch

Sequence alignment and phylogenetic analysis

Subcellular localization

RNA-seq analysis

Statistics and reproducibility

Declarations

References

Supplementary Files

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