CCCH Zinc Finger Proteins Research Articles

Overexpression of stress granule protein TZF1 enhances salt stress tolerance by targeting ACA11 mRNA for degradation in Arabidopsis.

Tandem CCCH zinc finger (TZF) proteins play diverse roles in plant growth and stress response. Although as many as 11 TZF proteins have been identified in Arabidopsis, little is known about the mechanism by which TZF proteins select and regulate the target mRNAs. Here, we report that Arabidopsis TZF1 is a bona-fide stress granule protein. Ectopic expression of TZF1 (TZF1 OE), but not an mRNA binding-defective mutant (TZF1H186Y OE), enhances salt stress tolerance in Arabidopsis. RNA-seq analyses of NaCl-treated plants revealed that the down-regulated genes in TZF1 OE plants are enriched for functions in salt and oxidative stress responses. Because many of these down-regulated mRNAs contain AU- and/or U-rich elements (AREs and/or UREs) in their 3'-UTRs, we hypothesized that TZF1-ARE/URE interaction might contribute to the observed gene expression changes. Results from RNA immunoprecipitation-quantitative PCR analysis, gel-shift, and mRNA half-life assays indicate that TZF1 binds and triggers degradation of the autoinhibited Ca2+-ATPase 11 (ACA11) mRNA, which encodes a tonoplast-localized calcium pump that extrudes calcium and dampens signal transduction pathways necessary for salt stress tolerance. Furthermore, this salt stress-tolerance phenotype was recapitulated in aca11 null mutants. Collectively, our findings demonstrate that TZF1 binds and initiates degradation of specific mRNAs to enhance salt stress tolerance.

Abstract 1086: ZFP36L2 is expected to be a potential prognostic marker in IL-1β+ osteosarcoma patients

Abstract Background: Osteosarcoma (OS) is the most common bone malignancy affect children and young adults. For decades, the long-term survival for patients with recurrences and metastases remains ~30%. There’s an urgent need for new prognostic marker and therapeutic options. ZFP36L2, a member of the tristetraprolin (TTP) family of CCCH zinc finger proteins, stands out for its pivotal role in post transcriptional modifications and modify tumor microenvironment (TME). Notably, ZFP36L2 contributes to the recruitment of macrophages, specifically the M1 subtype, which are known for their ability to secrete IL1β and exhibit anti-tumor functions. Our study reveals that, ZFP36L2 can be used as a predictor of prognosis in OS patients with enriched IL1B expression. This study aims to elucidate the interaction between ZFP36L2 and IL1B in modification of TME and shed light on its potential of prognosis prediction and therapeutic in IL1β positive OS patients. Methods: Firstly, we conducted a comprehensive exploration of ZFP36L2 expression across TCGA cohorts. Then, we performed IHC staining to confirm the evaluated expression of ZFP36L2 in cancerous tissues. Immune infiltration analysis, including QUANTISEQ, TIMER, and CIBERSORT, was conducted. KM plot was employed to analyze the prognosis of OS patients. Finally, qPCR and western blot were used to validate transcriptional and translational changes of key molecules across OS cell lines. Results: Our findings demonstrate that ZFP36L2 exhibits specific expression across various cancer types, with a notable elevation in sarcoma tissues, as confirmed by IHC. Importantly, this high expression of ZFP36L2 in sarcoma tissues is correlated with metastasis. We found ZFP36L2 significantly correlates with tumor metastasis but failed in patient overall survival. Immunoinfiltration analysis indicates a significant correlation between ZFP36L2 and macrophages, as well as T cells, suggesting a role in the TME. Prognostic analysis reveals that ZFP36L2 can serve as a predictive marker for IL1β positive patients. The wetlab experiments demonstrated that ZFP36L2 leads to IRGM mRNA degradation, ultimately affecting IL1β+ macrophages. Conclusion: This study delineates the tumor-specific elevated expression of ZFP36L2 and its potential as a prognostic marker in IL1β+ OS patients. We hypothesize that ZFP36L2 affects IL1β+ macrophages through IRGM degradation. Besides, IRGM also has an impact on cytotoxic T cells. Therefore, the ZFP36L2-IRGM axis plays an important role in remodeling OS TME. These results indicated ZFP36L2, not only serves as a marker, but also can be a therapeutic target in IL1β enriched patients. Citation Format: Peiyao Hao, Piaopiao Luo, Zhenhua Ren, Shenglin Xu, Jijun Han, Xiang Nan. ZFP36L2 is expected to be a potential prognostic marker in IL-1β+ osteosarcoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1086.

Ectopic expression of TTP gene from human in poplar promotes xylem differentiation and confers plant drought tolerance.

The CCCH zinc finger proteins play critical roles in a wide variety of growth, development, and stress responses. Currently, limited reports are available about the roles of animal CCCH proteins in plants. In this study, we report the identification of human TTP (hTTP) with functional similarity to PdC3H17 in a hybrid poplar. hTTP and PdC3H17 shared highly similar tandem CCCH zinc-finger RNA-binding domains. The fragments excluding the CCCH domain of both hTTP and PdC3H17 possessed transcriptional activation activities in yeast cells. Compared to the controls, ectopic expression of hTTP in poplar caused dwarfism, and resulted in significant increases in stem xylem vessel number and photosynthetic and ROS-scavenging abilities, thereby enhancing plant tolerance to drought stress. Our results suggest that hTTP may perform a function in poplar through the PdC3H17-mediated system, and provide an example for the application of animal genes in plants.

Heterologous Expression of Two Brassica campestris CCCH Zinc-Finger Proteins in Arabidopsis Induces Cytoplasmic Foci and Causes Pollen Abortion

The membrane-less organelles in cytoplasm that are presented as cytoplasmic foci were successively identified. Although multiple CCCH zinc-finger proteins have been found to be localized in cytoplasmic foci, the relationship between their specific localization and functions still needs further clarification. Here, we report that the heterologous expression of two Brassica campestris CCCH zinc-finger protein genes (BcMF30a and BcMF30c) in Arabidopsis thaliana can affect microgametogenesis by involving the formation of cytoplasmic foci. By monitoring the distribution of proteins and observing pollen phenotypes, we found that, when these two proteins were moderately expressed in pollen, they were mainly dispersed in the cytoplasm, and the pollen developed normally. However, high expression induced the assembly of cytoplasmic foci, leading to pollen abortion. These findings suggested that the continuous formation of BcMF30a/BcMF30c-associated cytoplasmic foci due to high expression was the inducement of male sterility. A co-localization analysis further showed that these two proteins can be recruited into two well-studied cytoplasmic foci, processing bodies (PBs), and stress granules (SGs), which were confirmed to function in mRNA metabolism. Together, our data suggested that BcMF30a and BcMF30c play component roles in the assembly of pollen cytoplasmic foci. Combined with our previous study on the homologous gene of BcMF30a/c in Arabidopsis, we concluded that the function of these homologous genes is conserved and that cytoplasmic foci containing BcMF30a/c may participate in the regulation of gene expression in pollen by regulating mRNA metabolism.

Transcriptome Analysis Reveals the Molecular Mechanism of Grape Inflorescence Elongation after Applying Microbial Fertilizers

Microbial fertilizers can activate and promote nutrient absorption and help inflorescence elongation. To understand the molecular mechanisms governing grape (Vitis vinifera) inflorescence elongation after microbial fertilizer application, we comprehensively analyzed the transcriptome dynamics of ‘Summer Black’ grape inflorescence at different leaf stages. With the development of ‘Summer Black’ grape inflorescence, gibberellic acid content gradually increased and was clearly higher in the microbial fertilizer group than in the corresponding control group. In addition, the microbial fertilizer and control groups had 291, 487, 490, 287, and 323 differentially expressed genes (DEGs) at the 4-, 6-, 8-, 10-, and 12-leaf stages, respectively. Kyoto Encyclopedia of Genes and Genomes pathway annotation revealed that most upregulated DEGs were enriched in starch and sucrose metabolism pathways at the 6-, 8-, and 10-leaf stages. Weighted gene coexpression network analysis identified stage-specific expression of most DEGs. In addition, multiple transcription factors and phytohormone signaling-related genes were found at different leaf stages, including basic helix-loop-helix proteins, CCCH zinc finger proteins, gibberellin receptor GID1A, 2-glycosyl hydrolases family 16, protein TIFY, MYB transcription factors, WRKY transcription factors, and ethylene response factor, suggesting that many transcription factors play important roles in inflorescence elongation at different developmental stages. These results provide valuable insights into the dynamic transcriptomic changes of inflorescence elongation at different leaf stages.

Hormone Regulation of CCCH Zinc Finger Proteins in Plants.

CCCH zinc finger proteins contain one to six tandem CCCH motifs composed of three cysteine and one histidine residues and have been widely found in eukaryotes. Plant CCCH proteins control a wide range of developmental and adaptive processes through DNA-protein, RNA-protein and/or protein-protein interactions. The complex networks underlying these processes regulated by plant CCCH proteins are often involved in phytohormones as signal molecules. In this review, we described the evolution of CCCH proteins from green algae to vascular plants and summarized the functions of plant CCCH proteins that are influenced by six major hormones, including abscisic acid, gibberellic acid, brassinosteroid, jasmonate, ethylene and auxin. We further compared the regulatory mechanisms of plant and animal CCCH proteins via hormone signaling. Among them, Arabidopsis AtC3H14, 15 and human hTTP, three typical CCCH proteins, are able to integrate multiple hormones to participate in various biological processes.

Transcriptome-Wide Identification of CCCH-Type Zinc Finger Proteins Family in Pinus massoniana and RR-TZF Proteins in Stress Response.

CCCH-type zinc finger proteins play an important role in multiple biotic and abiotic stresses. More and more reports about CCCH functions in plant development and stress responses have appeared over the past few years, focusing especially on tandem CCCH zinc finger proteins (TZFs). However, this has not been reported in Pinaceae. In this study, we identified 46 CCCH proteins, including 6 plant TZF members in Pinus massoniana, and performed bioinformatic analysis. According to RT-PCR analysis, we revealed the expression patterns of five RR-TZF genes under different abiotic stresses and hormone treatments. Meanwhile, tissue-specific expression analysis suggested that all genes were mainly expressed in needles. Additionally, RR-TZF genes showed transcriptional activation activity in yeast. The results in this study will be beneficial in improving the stress resistance of P. massoniana and facilitating further studies on the biological and molecular functions of CCCH zinc finger proteins.

Highly Overexpressed AtC3H18 Impairs Microgametogenesis via Promoting the Continuous Assembly of mRNP Granules.

Plant CCCH zinc-finger proteins form a large family of regulatory proteins function in many aspects of plant growth, development and environmental responses. Despite increasing reports indicate that many CCCH zinc-finger proteins exhibit similar subcellular localization of being localized in cytoplasmic foci, the underlying molecular mechanism and the connection between this specific localization pattern and protein functions remain largely elusive. Here, we identified another cytoplasmic foci-localized CCCH zinc-finger protein, AtC3H18, in Arabidopsis thaliana. AtC3H18 is predominantly expressed in developing pollen during microgametogenesis. Although atc3h18 mutants did not show any abnormal phenotype, possibly due to redundant gene(s), aberrant AtC3H18 expression levels caused by overexpression resulted in the assembly of AtC3H18-positive granules in a dose-dependent manner, which in turn led to male sterility phenotype, highlighting the importance of fine-tuned AtC3H18 expression. Further analyzes demonstrated that AtC3H18-positive granules are messenger ribonucleoprotein (mRNP) granules, since they can exhibit liquid-like physical properties, and are associated with another two mRNP granules known as processing bodies (PBs) and stress granules (SGs), reservoirs of translationally inhibited mRNAs. Moreover, the assembly of AtC3H18-positive granules depends on mRNA availability. Combined with our previous findings on the AtC3H18 homologous genes in Brassica campestris, we concluded that appropriate expression level of AtC3H18 during microgametogenesis is essential for normal pollen development, and we also speculated that AtC3H18 may act as a key component of mRNP granules to modulate pollen mRNAs by regulating the assembly/disassembly of mRNP granules, thereby affecting pollen development.

The Plasmodium falciparum CCCH Zinc Finger Protein ZNF4 Plays an Important Role in Gametocyte Exflagellation through the Regulation of Male Enriched Transcripts.

CCCH zinc finger proteins (ZFPs) function mainly as RNA-binding proteins (RBPs) and play a central role in the mRNA metabolism. Over twenty seven CCCH-ZFPs are encoded in the genome of the human malaria parasite Plasmodium falciparum, the causative agent of malaria tropica. However, little is known about their functions. In this study, we characterize one member of the PfCCCH-ZFP named ZNF4. We show that ZNF4 is highly expressed in mature gametocytes, where it predominantly localizes to the cytoplasm. Targeted gene disruption of ZNF4 showed no significant effect in asexual blood stage replication and gametocyte development while male gametocyte exflagellation was significantly impaired, leading to reduced malaria transmission in the mosquito. Comparative transcriptomics between wildtype (WT) and the ZNF4-deficient line (ZNF4-KO) demonstrated the deregulation of about 473 genes (274 upregulated and 199 downregulated) in mature gametocytes. Most of the downregulated genes show peak expression in mature gametocyte with male enriched genes associated to the axonemal dynein complex formation, and cell projection organization is highly affected, pointing to the phenotype in male gametocyte exflagellation. Upregulated genes are associated to ATP synthesis. Our combined data therefore indicate that ZNF4 is a CCCH zinc finger protein which plays an important role in male gametocyte exflagellation through the regulation of male gametocyte-enriched genes.

RNA Binding Protein OsTZF7 Traffics Between the Nucleus and Processing Bodies/Stress Granules and Positively Regulates Drought Stress in Rice.

Tandem CCCH zinc finger (TZF) proteins are the essential components of processing bodies (PBs) and stress granules (SGs), which play critical roles in growth development and stress response in both animals and plants through posttranscriptional regulation of target mRNA. In this study, we characterized the biological and molecular functions of a novel tandem zinc finger protein, OsTZF7. The expression of OsTZF7 was upregulated by abiotic stresses, including polyethylene glycol (PEG) 4000, NaCl, and abscisic acid (ABA) in rice. Accordingly, the overexpression of OsTZF7 increased drought tolerance and enhanced sensitivity to exogenous ABA in rice, whereas the knockdown of OsTZF7 resulted in the opposite phenotype. RNA-seq analysis revealed that genes related to “response to stress,” “abscisic acid signaling,” “methylated histone binding,” and “cytoplasmic mRNA processing body” are regulated by OsTZF7. We demonstrated that OsTZF7 can traffic between the nucleus and PBs/SGs, and the leucine-rich nuclear export signal (NES) mediates the nuclear export of OsTZF7. Additionally, we revealed that OsTZF7 can bind adenine- and uridine-rich (AU-rich) element (ARE) or ARE-like motifs within the 3′ untranslated region of downregulated mRNAs, and interact with PWWP family proteins in vitro. Together, these results indicate that OsTZF7 positively regulates drought response in rice via ABA signaling and may be involved in mRNA turnover.

Non-TZF Transcriptional Activator AtC3H12 Negatively Affects Seed Germination and Seedling Development in Arabidopsis.

CCCH zinc finger proteins are a large protein family and are classified as either tandem CCCH zinc finger (TZF) or non-TZF proteins. The roles of TZF genes in several plants have been well determined, whereas the functions of many non-TZF genes in plants remain uncharacterized. Herein, we describe biological and molecular functions of AtC3H12, an Arabidopsis non-TZF protein containing three CCCH zinc finger motifs. AtC3H12 has orthologs in several plant species but has no paralog in Arabidopsis. AtC3H12-overexpressing transgenic plants (OXs) germinated slower than wild-type (WT) plants, whereas atc3h12 mutants germinated faster than WT plants. The fresh weight (FW) and primary root lengths of AtC3H12 OX seedlings were lighter and shorter than those of WT seedlings, respectively. In contrast, FW and primary root lengths of atc3h12 seedlings were heavier and longer than those of WT seedlings, respectively. AtC3H12 was localized in the nucleus and displayed transactivation activity in both yeast and Arabidopsis. We found that the 97–197 aa region of AtC3H12 is an important part for its transactivation activity. Detection of expression levels and analysis of Arabidopsis transgenic plants harboring a PAtC3H12::GUS construct showed that AtC3H12 expression increases as the Arabidopsis seedlings develop. Taken together, our results demonstrate that AtC3H12 negatively affects seed germination and seedling development as a nuclear transcriptional activator in Arabidopsis. To our knowledge, this is the first report to show that non-TZF proteins negatively affect plant development as nuclear transcriptional activators.

The APC/CFZY-1/Cdc20 Complex Coordinates With OMA-1 to Regulate the Oocyte-to-Embryo Transition in Caenorhabditis elegans.

During oocyte maturation and the oocyte-to-embryo transition, key developmental regulators such as RNA-binding proteins coordinate translation of particular messenger RNA (mRNAs) and related developmental processes by binding to their cognate maternal mRNAs. In the nematode Caenorhabditis elegans, these processes are regulated by a set of CCCH zinc finger proteins. Oocyte maturation defective-1 (OMA-1) and OMA-2 are two functionally redundant CCCH zinc finger proteins that turnover rapidly during the first embryonic cell division. These turnovers are required for proper transition from oogenesis to embryogenesis. A gain-of-function mutant of OMA-1, oma-1(zu405), stabilizes and delays degradation of OMA-1, resulting in delayed turnover and mis-segregation of other cell fate determinants, which eventually causes embryonic lethality. We performed a large-scale forward genetic screen to identify suppressors of the oma-1(zu405) mutant. We show here that multiple alleles affecting functions of various anaphase promoting complex/cyclosome (APC/C) subunits, including MAT-1, MAT-2, MAT-3, EMB-30, and FZY-1, suppress the gain-of-function mutant of OMA-1. Transcriptome analysis suggested that overall transcription in early embryos occurred after introducing mutations in APC/C genes into the oma-1(zu405) mutant. Mutations in APC/C genes prevent OMA-1 enrichment in P granules and correct delayed degradation of downstream cell fate determinants including pharynx and intestine in excess-1 (PIE-1), posterior segregation-1 (POS-1), muscle excess-3 (MEX-3), and maternal effect germ-cell defective-1 (MEG-1). We demonstrated that only the activator FZY-1, but not FZR-1, is incorporated in the APC/C complex to regulate the oocyte-to-embryo transition. Our findings suggested a genetic relationship linking the APC/C complex and OMA-1, and support a model in which the APC/C complex promotes P granule accumulation and modifies RNA binding of OMA-1 to regulate the oocyte-to-embryo transition process.

The Roles of CCCH Zinc-Finger Proteins in Plant Abiotic Stress Tolerance.

Zinc-finger proteins, a superfamily of proteins with a typical structural domain that coordinates a zinc ion and binds nucleic acids, participate in the regulation of growth, development, and stress adaptation in plants. Most zinc fingers are C2H2-type or CCCC-type, named after the configuration of cysteine (C) and histidine (H); the less-common CCCH zinc-finger proteins are important in the regulation of plant stress responses. In this review, we introduce the domain structures, classification, and subcellular localization of CCCH zinc-finger proteins in plants and discuss their functions in transcriptional and post-transcriptional regulation via interactions with DNA, RNA, and other proteins. We describe the functions of CCCH zinc-finger proteins in plant development and tolerance to abiotic stresses such as salt, drought, flooding, cold temperatures and oxidative stress. Finally, we summarize the signal transduction pathways and regulatory networks of CCCH zinc-finger proteins in their responses to abiotic stress. CCCH zinc-finger proteins regulate the adaptation of plants to abiotic stress in various ways, but the specific molecular mechanisms need to be further explored, along with other mechanisms such as cytoplasm-to-nucleus shuttling and post-transcriptional regulation. Unraveling the molecular mechanisms by which CCCH zinc-finger proteins improve stress tolerance will facilitate the breeding and genetic engineering of crops with improved traits.

The RNase MCPIP3 promotes skin inflammation by orchestrating myeloid cytokine response

CCCH zinc finger proteins resolve immune responses by degrading the mRNAs of inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin (IL)-6. Here we report that one such family member, monocyte chemotactic protein-induced protein 3 (MCPIP3, also named ZC3H12C or Regnase-3), promotes skin inflammation by simultaneously enhancing TNF in macrophages and repressing IL-6 in plasmacytoid dendritic cells (pDCs). MCPIP3 is positively associated with psoriasis pathogenesis, and highly expressed by macrophages and pDCs. MCPIP3-deficient macrophages produce less TNF and IL-12p40. However, MCPIP3-deficient pDCs secrete significantly more IL-6. This enhanced intradermal IL-6 may alleviate imiquimod-induced skin inflammation. As a result, MCPIP3-deficient mice are protected from imiquimod-induced psoriasiform lesions. Furthermore, early exposure to pDC-derived IL-6 suppresses macrophage-derived TNF and IL-12p40. Mechanistically, MCPIP3 could directly degrade mRNAs of IL-6, Regnase-1, and IκBζ. In turn, Regnase-1 could degrade MCPIP3 mRNAs. Our study identifies a critical post-transcriptional mechanism that synchronizes myeloid cytokine secretion to initiate autoimmune skin inflammation.

Non-TZF Protein AtC3H59/ZFWD3 Is Involved in Seed Germination, Seedling Development, and Seed Development, Interacting with PPPDE Family Protein Desi1 in Arabidopsis

Despite increasing reports on the function of CCCH zinc finger proteins in plant development and stress response, the functions and molecular aspects of many non-tandem CCCH zinc finger (non-TZF) proteins remain uncharacterized. AtC3H59/ZFWD3 is an Arabidopsis non-TZF protein and belongs to the ZFWD subfamily harboring a CCCH zinc finger motif and a WD40 domain. In this study, we characterized the biological and molecular functions of AtC3H59, which is subcellularly localized in the nucleus. The seeds of AtC3H59-overexpressing transgenic plants (OXs) germinated faster than those of wild type (WT), whereas atc3h59 mutant seeds germinated slower than WT seeds. AtC3H59 OX seedlings were larger and heavier than WT seedlings, whereas atc3h59 mutant seedlings were smaller and lighter than WT seedlings. Moreover, AtC3H59 OX seedlings had longer primary root length than WT seedlings, whereas atc3h59 mutant seedlings had shorter primary root length than WT seedlings, owing to altered cell division activity in the root meristem. During seed development, AtC3H59 OXs formed larger and heavier seeds than WT. Using yeast two-hybrid screening, we isolated Desi1, a PPPDE family protein, as an interacting partner of AtC3H59. AtC3H59 and Desi1 interacted via their WD40 domain and C-terminal region, respectively, in the nucleus. Taken together, our results indicate that AtC3H59 has pleiotropic effects on seed germination, seedling development, and seed development, and interacts with Desi1 in the nucleus via its entire WD40 domain. To our knowledge, this is the first report to describe the biological functions of the ZFWD protein and Desi1 in Arabidopsis.

Identification of CCCH Zinc Finger Proteins Family in Moso Bamboo (Phyllostachys edulis), and PeC3H74 Confers Drought Tolerance to Transgenic Plants.

CCCH zinc finger proteins are a class of important zinc-finger transcription factors and have functions in various plant growth and stress responses, but their functions in moso bamboo (Phyllostachys edulis) are unclear. In this current study, we main investigated the structures, phylogenetic relationships, promoter elements and microsynteny of PeC3Hs. In this research, 119 CCCH zinc finger proteins (PeC3H1-119) identified genes in moso bamboo were divided into 13 subfamilies (A-M) based on phylogenetic analysis. Meanwhile, moso bamboo were treated with abscisic acid (ABA), methyl jasmonate (Me-JA) and gibberellic acid (GA) and 12 CCCH genes expression levels were assayed using qRT-PCR. In the three hormone treatments, 12 genes were up-regulated or down-regulated, respectively. In addition, PeC3H74 was localized on the cytomembrane, and it had self-activation activities. Phenotypic and physiological analysis showed that PeC3H74 (PeC3H74-OE) conferred drought tolerance of transgenic Arabidopsis, including H2O2 content, survival rate, electrolyte leakage as well as malondialdehyde content. Additionally, compared with wild-type plants, transgenic Arabidopsis thaliana seedling roots growth developed better under 10 μM ABA; Moreover, the stomatal of over-expressing PeC3H74 in Arabidopsis changed significantly under ABA treatment. The above results suggest that PeC3H74 was quickly screened by bioinformatics, and it may enhanced drought tolerance in plants through the ABA-dependent signaling pathway.

Overexpression of OsC3H10, a CCCH-Zinc Finger, Improves Drought Tolerance in Rice by Regulating Stress-Related Genes.

CCCH zinc finger proteins are members of the zinc finger protein family, and are known to participate in the regulation of development and stress responses via the posttranscriptional regulation of messenger RNA in animals and yeast. However, the molecular mechanism of CCCHZF-mediated drought tolerance is not well understood. We analyzed the functions of OsC3H10, a member of the rice CCCHZF family. OsC3H10 is predominantly expressed in seeds, and its expression levels rapidly declined during seed imbibition. The expression of OsC3H10 was induced by drought, high salinity and abscisic acid (ABA). Subcellular localization analysis revealed that OsC3H10 localized not only in the nucleus but also to the processing bodies and stress granules upon stress treatment. Root-specific overexpression of OsC3H10 was insufficient to induce drought tolerance, while the overexpression of OsC3H10 throughout the entire plant enhanced the drought tolerance of rice plants. Transcriptome analysis revealed that OsC3H10 overexpression elevated the expression levels of genes involved in stress responses, including LATE EMBRYOGENESIS ABUNDANT PROTEINs (LEAs), PATHOGENESIS RELATED GENEs (PRs) and GERMIN-LIKE PROTEINs (GLPs). Our results demonstrated that OsC3H10 is involved in the regulation of the drought tolerance pathway by modulating the expression of stress-related genes.

BcMF30a and BcMF30c, Two Novel Non-Tandem CCCH Zinc-Finger Proteins, Function in Pollen Development and Pollen Germination in Brassica campestris ssp. chinensis

Chinese cabbage (Brassica campestris) is an economically important leaf vegetable crop worldwide. Mounting studies have shown that cysteine-cysteine-cysteine-histidine (CCCH) zinc-finger protein genes are involved in various plant growth and development processes. However, research on the involvement of these genes in male reproductive development is still in its infancy. Here, we identified 11 male fertility-related CCCH genes in Chinese cabbage. Among them, a pair of paralogs encoding novel non-tandem CCCH zinc-finger proteins, Brassica campestris Male Fertility 30a (BcMF30a) and BcMF30c, were further characterized. They were highly expressed in pollen during microgametogenesis and continued to express in germinated pollen. Further analyses demonstrated that both BcMF30a and BcMF30c may play a dual role as transcription factors and RNA-binding proteins in plant cells. Functional analysis showed that partial bcmf30a bcmf30c pollen grains were aborted due to the degradation of pollen inclusion at the microgametogenesis phase, and the germination rate of viable pollen was also greatly reduced, indicating that BcMF30a and BcMF30c are required for both pollen development and pollen germination. This research provided insights into the function of CCCH proteins in regulating male reproductive development and laid a theoretical basis for hybrid breeding of Chinese cabbage.

Overexpression of PdC3H17 Confers Tolerance to Drought Stress Depending on Its CCCH Domain in Populus.

Plant CCCH zinc finger proteins control growth, development, and stress responses mainly at the post-transcriptional level. Currently, limited reports are available about the roles of plant CCCH proteins in drought tolerance. In this study, we provided evidence showing that PdC3H17 from Populus deltoides × P. euramericana involves drought tolerance and response. Overexpression of PdC3H17 in poplar caused dwarf, resulted in higher stem water potential, and showed increased photosynthetic and ROS-scavenging abilities, thereby enhancing tolerance to drought stress, compared to controls. Accordingly, after drought treatment the stem elongation and thickening rates of these overexpression lines were higher than those of the controls. However, overexpression of the coding region excluding the CCCH domain of PdC3H17 roughly exhibited WT-like physiological and drought-resistant phenotypes, indicating the requirement of the CCCH domain for PdC3H17 controlling these processes. In addition, N-terminal sequence of PdC3H17 was found to possess transcriptional activity ability in yeast cells. Together, our results suggest that PdC3H17 may depend on its CCCH domain to control drought tolerance in Populus.

CaC3H14 encoding a tandem CCCH zinc finger protein is directly targeted by CaWRKY40 and positively regulates the response of pepper to inoculation by Ralstonia solanacearum.

Tandem CCCH zinc finger (TZnF) proteins have been implicated in plant defence, but their role in pepper (Capsicum annuum) is unclear. In the present study, the role of CaC3H14, a pepper TZnF protein, in the immune response of pepper plants to Ralstonia solanacearum infection was characterized. When fused to the green fluorescent protein, CaC3H14 was localized exclusively to the nuclei in leaf cells of Nicotiana benthamiana plants transiently overexpressing CaC3H14. Transcript abundance of CaC3H14 was up-regulated by inoculation with R.solanacearum. Virus-induced silencing of CaC3H14 increased the susceptibility of the plants to R.solanacearum and down-regulated the genes associated with the hypersensitive response (HR), specifically HIR1 and salicylic acid (SA)-dependent PR1a. By contrast, silencing resulted in the up-regulation of jasmonic acid (JA)-dependent DEF1 and ethylene (ET) biosynthesis-associated ACO1. Transient overexpression of CaC3H14 in pepper triggered an intensive HR, indicated by cell death and hydrogen peroxide (H2 O2 ) accumulation, up-regulated PR1a and down-regulated DEF1 and ACO1. Ectopic overexpression of CaC3H14 in tobacco plants significantly decreased the susceptibility of tobacco plants to R.solanacearum. It also up-regulated HR-associated HSR515, immunity-associated GST1 and the SA-dependent marker genes NPR1 and PR2, but down-regulated JA-dependent PR1b and ET-dependent EFE26. The CaC3H14 promoter and was bound and its transcription was up-regulated by CaWRKY40. Collectively, these results indicate that CaC3H14 is transcriptionally targeted by CaWRKY40, is a modulator of the antagonistic interaction between SA and JA/ET signalling, and enhances the defence response of pepper plants to infection by R.solanacearum.