C2H2 Zinc Finger Protein Research Articles

Heterologous expression of Zygophyllum xanthoxylon zinc finger protein gene (ZxZF) enhances the tolerance of poplar photosynthetic function to drought stress

The ZxZF transcription factor (TF) of Zygophyllum xanthoxylon (Bunge) Maxim, an extremely drought-resistant woody plant, is a C2H2 zinc finger protein. Studies have shown that C2H2 zinc finger proteins play important roles in activating stress-related genes and enhancing plant resistance. However, their function in regulating plant photosynthesis under drought stress is not well understood. Since poplar is an important greening and afforestation tree species, it is particularly important to cultivate excellent drought-tolerant varieties. The ZxZF transcription factor (TF) was heterogeneously expressed in Euroamerican poplar (Populus × euroameracana cl.'Bofengl’) by genetic transformation. Based on the mechanism and potential function of poplar photosynthesis regulated by ZxZF under drought stress, transcriptomic and physiological determinations were used to reveal the important role of this gene in improving the drought resistance of poplar. The results showed that the overexpression of ZxZF TF in transgenic poplars could improve the inhibition of Calvin cycle by regulating stomatal opening and increasing the concentration of intercellular CO2. The chlorophyll content, photosynthetic performance index, and photochemical efficiency of transgenic lines under drought stress were significantly higher than those of the wild type (WT). The overexpression of ZxZF TFs could alleviate the degree of photoinhibition of photosystems II and I under drought stress and maintain the efficiency of light energy capture and the photosynthetic electron transport chain. The transcriptomic data also showed that differentially expressed genes between the transgenic poplar and WT under drought stress were primarily enriched in metabolic pathways related to photosynthesis, such as photosynthesis, photosynthesis-antenna protein, porphyrin and chlorophyll metabolism, and photosynthetic carbon fixation, and the downregulation of genes related to chlorophyll synthesis, photosynthetic electron transport and Calvin cycle were alleviated. In addition, the overexpression of ZxZF TF can alleviate the inhibition of NADH dehydrogenase-like (NDH) cyclic electron flow of the poplar NDH pathway under drought stress, which plays an important role in reducing the excess pressure of electrons on the photosynthetic electron transport chain and maintaining the normal photosynthetic electron transport. In summary, the overexpression of ZxZF TFs can effectively alleviate the inhibition of drought on the assimilation of carbon in poplar and have a positive impact on light energy capture, the orderly transport of photosynthetic electron transport chain and the integrity of the photosystem, which is highly significant to acheivean in-depth understanding of the function of ZxZF TFs. This also provides an important basis for the breeding of new transgenic poplar varieties.

The nuclear effector MoHTR3 of Magnaporthe oryzae modulates host defence signalling in the biotrophic stage of rice infection.

Fungal effectors play a pivotal role in suppressing the host defence system, and their evolution is highly dynamic. By comparative sequence analysis of plant-pathogenic fungi and Magnaporthe oryzae, we identified the small secreted C2 H2 zinc finger protein MoHTR3. MoHTR3 exhibited high conservation in M. oryzae strains but low conservation among other plant-pathogenic fungi, suggesting an emerging evolutionary selection process. MoHTR3 is exclusively expressed in the biotrophic stage of fungal invasion, and the encoded protein localizes to the biotrophic interfacial complex (BIC) and the host cell nucleus. The signal peptide crucial for MoHTR3' secretion to the BIC and the protein section required for its translocation to the nucleus were both identified by a functional protein domain study. The host-nuclear localization of MoHTR3 suggests a function as a transcriptional modulator of host defence gene induction. After ΔMohtr3 infection, the expression of jasmonic acid- and ethylene-associated genes was diminished in rice, in contrast to when the MoHTR3-overexpressing strain (MoHTR3ox) was applied. The transcript levels of salicylic acid- and defence-related genes were also affected after ΔMohtr3 and MoHTR3ox application. In pathogenicity assays, ΔMohtr3 was indistinguishable from the wild type. However, MoHTR3ox-infected plants showed diminished lesion formation and hydrogen peroxide accumulation, accompanied by a decrease in susceptibility, suggesting that the MoHTR3-induced manipulation of host cells affects host-pathogen interaction. MoHTR3 emphasizes the role of the host nucleus as a critical target for the pathogen-driven manipulation of host defence mechanisms and underscores the ongoing evolution of rice blast's arms race.

Comprehensive Analysis of the INDETERMINATE DOMAIN (IDD) Gene Family and Their Response to Abiotic Stress in Zea mays

Transcription factors (TFs) are important regulators of numerous gene expressions due to their ability to recognize and combine cis-elements in the promoters of target genes. The INDETERMINATE DOMAIN (IDD) gene family belongs to a subfamily of C2H2 zinc finger proteins and has been identified only in terrestrial plants. Nevertheless, little study has been reported concerning the genome-wide analysis of the IDD gene family in maize. In total, 22 ZmIDD genes were identified, which can be distributed on 8 chromosomes in maize. On the basis of evolutionary relationships and conserved motif analysis, ZmIDDs were categorized into three clades (1, 2, and 3), each owning 4, 6, and 12 genes, respectively. We analyzed the characteristics of gene structure and found that 3 of the 22 ZmIDD genes do not contain an intron. Cis-element analysis of the ZmIDD promoter showed that most ZmIDD genes possessed at least one ABRE or MBS cis-element, and some ZmIDD genes owned the AuxRR-core, TCA-element, TC-rich repeats, and LTR cis-element. The Ka:Ks ratio of eight segmentally duplicated gene pairs demonstrated that the ZmIDD gene families had undergone a purifying selection. Then, the transcription levels of ZmIDDs were analyzed, and they showed great differences in diverse tissues as well as abiotic stresses. Furthermore, regulatory networks were constructed through the prediction of ZmIDD-targeted genes and miRNAs, which can inhibit the transcription of ZmIDDs. In total, 6 ZmIDDs and 22 miRNAs were discovered, which can target 180 genes and depress the expression of 9 ZmIDDs, respectively. Taken together, the results give us valuable information for studying the function of ZmIDDs involved in plant development and climate resilience in maize.

On the dependent recognition of some long zinc finger proteins.

The human genome contains about 800 C2H2 zinc finger proteins (ZFPs), and most of them are composed of long arrays of zinc fingers. Standard ZFP recognition model asserts longer finger arrays should recognize longer DNA-binding sites. However, recent experimental efforts to identify in vivo ZFP binding sites contradict this assumption, with many exhibiting short motifs. Here we use ZFY, CTCF, ZIM3, and ZNF343 as examples to address three closely related questions: What are the reasons that impede current motif discovery methods? What are the functions of those seemingly unused fingers and how can we improve the motif discovery algorithms based on long ZFPs' biophysical properties? Using ZFY, we employed a variety of methods and find evidence for 'dependent recognition' where downstream fingers can recognize some previously undiscovered motifs only in the presence of an intact core site. For CTCF, high-throughput measurements revealed its upstream specificity profile depends on the strength of its core. Moreover, the binding strength of the upstream site modulates CTCF's sensitivity to different epigenetic modifications within the core, providing new insight into how the previously identified intellectual disability-causing and cancer-related mutant R567W disrupts upstream recognition and deregulates the epigenetic control by CTCF. Our results establish that, because of irregular motif structures, variable spacing and dependent recognition between sub-motifs, the specificities of long ZFPs are significantly underestimated, so we developed an algorithm, ModeMap, to infer the motifs and recognition models of ZIM3 and ZNF343, which facilitates high-confidence identification of specific binding sites, including repeats-derived elements. With revised concept, technique, and algorithm, we can discover the overlooked specificities and functions of those 'extra' fingers, and therefore decipher their broader roles in human biology and diseases.

Identification and expression analysis of C2H2 zinc finger protein genes reveals their role in stress tolerance in Brassica napus.

C2H2-zinc finger genes are involved in various biological processes in plants including stress response; however, they lack characterization in Brassica napus. We identified 267 C2H2-ZF genes in B. napus and deciphered their physiological properties, subcellular localization, structure, synteny and phylogeny, and investigated expression of 20 genes in response to different stresses and phytohormone treatments. The 267 genes were distributed on 19 chromosomes; phylogenetic analysis placed them into five clades. They varied from 0.41 to 9.2 kb in length, had stress responsive cis-acting elements in promoter regions, and their protein length varied from 9 to 1,366 amino acids. About 42% of the genes had one exon, and 88% genes had orthologues in Arabidopsis thaliana. About 97% of the genes were located in nucleus and 3% in cytoplasmic organelles. qRT-PCR analysis showed different expression pattern of these genes in response to biotic stresses (Plasmodiophora brassicae and Sclerotinia sclerotiorum) and abiotic stresses (cold, drought and salinity) and hormonal treatments. Differential expression of the same gene was observed under multiple stress conditions, and a few genes showed similar expression in response to more than one phytohormones. Our results suggest that the C2H2-ZF genes can be targeted for the improvement of stress tolerance in canola.

Repressive ZINC FINGER OF ARABIDOPSIS THALIANA proteins promote programmed cell death in the Arabidopsis columella root cap.

Developmental programmed cell death (dPCD) controls a plethora of functions in plant growth and reproduction. In the root cap of Arabidopsis (Arabidopsis thaliana), dPCD functions to control organ size in balance with the continuous stem cell activity in the root meristem. Key regulators of root cap dPCD including SOMBRERO/ANAC033 (SMB) belong to the NAC family of transcription factors. Here, we identify the C2H2 zinc finger protein ZINC FINGER OF ARABIDOPSIS THALIANA 14 ZAT14 as part of the gene regulatory network of root cap dPCD acting downstream of SMB. Similar to SMB, ZAT14-inducible misexpression leads to extensive ectopic cell death. Both the canonical EAR motif and a conserved L-box motif of ZAT14 act as transcriptional repression motifs and are required to trigger cell death. While a single zat14 mutant does not show a cell death-related phenotype, a quintuple mutant knocking out 5 related ZAT paralogs shows a delayed onset of dPCD execution in the columella and the adjacent lateral root cap. While ZAT14 is co-expressed with established dPCD-associated genes, it does not activate their expression. Our results suggest that ZAT14 acts as a transcriptional repressor controlling a so far uncharacterized subsection of the dPCD gene regulatory network active in specific root cap tissues.

A C2H2 zinc finger transcription factor of the whitefly Bemisia tabaci interacts with the capsid proteins of begomoviruses and inhibits virus retention.

Begomoviruses are a group of ssDNA viruses exclusively transmitted by the whitefly Bemisia tabaci and constrain vegetable production in the old and new worlds. Although multiple molecular determinants governing the transmission of begomoviruses by whiteflies have been unravelled, factors critical for transmission majorly remain unknown. In this study, a whitefly C2H2 zinc finger (ZF) protein, 100% identical to the vascular endothelial ZF-like gene (vezf) protein was confirmed to interact with the CP of both old- and new-world begomoviruses. This was achieved by a yeast two-hybrid (Y2H) system screening of a whitefly cDNA library using capsid protein (CP) of TYLCV as a bait. In silico annotation of vezf protein revealed that it contains a N-terminal ZF-associated domain (ZAD) alongside multiple C2H2 ZF domains on the C-terminal end. ZAD-ZF proteins form the most abundant class of transcription factors within insects. Herein, we validated the interaction of vezf with four diverse begomoviruses and its functional role in begomovirus transmission. Silencing of the vezf gene of B. tabaci led to increased retention of three diverse begomoviruses tested. Vezf is the first insect transcription factor identified to interact with plant viruses and can be crucial to understand the possible mechanisms by which plant viruses modulate transcription of their insect vectors during transmission.

Application of an Improved 2-Dimensional High-Throughput Soybean Root Phenotyping Platform to Identify Novel Genetic Variants Regulating Root Architecture Traits.

Nutrient-efficient root system architecture (RSA) is becoming an important breeding objective for generating crop varieties with improved nutrient and water acquisition efficiency. Genetic variants shaping soybean RSA is key in improving nutrient and water acquisition. Here, we report on the use of an improved 2-dimensional high-throughput root phenotyping platform that minimizes background noise by imaging pouch-grown root systems submerged in water. We also developed a background image cleaning Python pipeline that computationally removes images of small pieces of debris and filter paper fibers, which can be erroneously quantified as root tips. This platform was used to phenotype root traits in 286 soybean lines genotyped with 5.4 million single-nucleotide polymorphisms. There was a substantially higher correlation in manually counted number of root tips with computationally quantified root tips (95% correlation), when the background was cleaned of nonroot materials compared to root images without the background corrected (79%). Improvements in our RSA phenotyping pipeline significantly reduced overestimation of the root traits influenced by the number of root tips. Genome-wide association studies conducted on the root phenotypic data and quantitative gene expression analysis of candidate genes resulted in the identification of 3 putative positive regulators of root system depth, total root length and surface area, and root system volume and surface area of thicker roots (DOF1-like zinc finger transcription factor, protein of unknown function, and C2H2 zinc finger protein). We also identified a putative negative regulator (gibberellin 20 oxidase 3) of the total number of lateral roots.

Analysis of the C2H2 Gene Family in Maize (Zea mays L.) under Cold Stress: Identification and Expression.

The C2H2 zinc finger protein is one of the most common zinc finger proteins, widely exists in eukaryotes, and plays an important role in plant growth and development, as well as in salt, low-temperature, and drought stress and other abiotic stress responses. In this study, C2H2 members were identified and analyzed from the low-temperature tolerant transcriptome sequencing data of maize seedlings. The chromosome position, physical and chemical properties, evolution analysis, gene structure, conservative motifs, promoter cis elements and collinearity relationships of gene the family members were analyzed using bioinformatics, and the expression of the ZmC2H2 gene family under cold stress was analyzed by fluorescent quantitative PCR. The results showed that 150 members of the C2H2 zinc finger protein family were identified, and their protein lengths ranged from 102 to 1223 bp. The maximum molecular weight of the ZmC2H2s was 135,196.34, and the minimum was 10,823.86. The isoelectric point of the ZmC2H2s was between 33.21 and 94.1, and the aliphatic index was 42.07-87.62. The promoter cis element analysis showed that the ZmC2H2 family contains many light-response elements, plant hormone-response elements, and stress-response elements. The analysis of the transcriptome data showed that most of the ZmC2H2 genes responded to cold stress, and most of the ZmC2H2 genes were highly expressed in cold-tolerant materials and lowly expressed in cold-sensitive materials. The real-time quantitative PCR (qRT-PCR) analysis showed that ZmC2H2-69, ZmC2H2-130, and ZmC2H2-76 were significantly upregulated, and that ZmC2H2-149, ZmC2H2-33, and ZmC2H2-38 were significantly downregulated. It is hypothesized that these genes, which function in different metabolic pathways, may play a key role in the maize cold response. These genes could be further studied as candidate genes. This study provides a theoretical reference for further study on the function analysis of the maize C2H2 gene family.

A 5'-Flanking C/G Pair at the Core Region Enhances the Recognition and Binding of Kaiso to Methylated DNA.

Methyl CpG binding proteins (MBPs) are transcription factors that recognize the methylated CpG sites in DNA and mediate the DNA methylation signal into various downstream cellular processes. The C2H2 zinc finger (ZF) protein, Kaiso, also an MBP, preferentially binds to two symmetrically methylated CpG sites in DNA sequences via C-terminal C2H2 ZF domains and mediates the transcription regulation process. Investigation of the molecular mechanism of the recognition of methylated DNA (meDNA) by Kaiso is important to understand how this protein reads and translates this methylation signal into downstream transcription outcomes. Despite previous studies in Kaiso-meDNA interactions, detailed structural investigations on the sequence-specific interaction of Kaiso with the meDNA sequence are still lacking. In this work, we used molecular modeling and molecular dynamics (MD) simulation-based computational approaches to investigate the recognition of various methylated DNA sequences by Kaiso. Our MD simulation results show that the Kaiso-meDNA interaction is sequence specific. The recognition of meDNA by Kaiso is enhanced in the MeECad sequence compared to the MeCG2 sequence. Compared to the 5'-flanking T/A pair in MeCG2, both MeCG2_mutCG and MeECad sequences show that a C/G base pair allows GLU535 of Kaiso to preferably recognize and bind the core mCpG site. The core mCGmCG site is crucial for the recognition process and formation of a stable complex. Our results reveal that the 5'-flanking nucleotides are also important for the enhanced binding and recognition of methylated sites.

Safeguarding Drosophila female germ cell identity depends on an H3K9me3 mini domain guided by a ZAD zinc finger protein.

H3K9me3-based gene silencing is a conserved strategy for securing cell fate, but the mechanisms controlling lineage-specific installation of this epigenetic mark remain unclear. In Drosophila, H3K9 methylation plays an essential role in securing female germ cell fate by silencing lineage inappropriate phf7 transcription. Thus, phf7 regulation in the female germline provides a powerful system to dissect the molecular mechanism underlying H3K9me3 deposition onto protein coding genes. Here we used genetic studies to identify the essential cis-regulatory elements, finding that the sequences required for H3K9me3 deposition are conserved across Drosophila species. Transposable elements are also silenced by an H3K9me3-mediated mechanism. But our finding that phf7 regulation does not require the dedicated piRNA pathway components, piwi, aub, rhino, panx, and nxf2, indicates that the mechanisms of H3K9me3 recruitment are distinct. Lastly, we discovered that an uncharacterized member of the zinc finger associated domain (ZAD) containing C2H2 zinc finger protein family, IDENTITY CRISIS (IDC; CG4936), is necessary for H3K9me3 deposition onto phf7. Loss of idc in germ cells interferes with phf7 transcriptional regulation and H3K9me3 deposition, resulting in ectopic PHF7 protein expression. IDC's role is likely to be direct, as it localizes to a conserved domain within the phf7 gene. Collectively, our findings support a model in which IDC guides sequence-specific establishment of an H3K9me3 mini domain, thereby preventing accidental female-to-male programming.

Zinc finger protein 251 deficiency impairs glucose metabolism by inducing adipocyte hypertrophy

Zinc finger protein (ZFP) 251 is a member of the C2H2 ZFP family containing a Krüppel-associated box domain that might mainly act as a transcriptional repressor. However, its cellular function remains largely unknown. Here, we discovered that ZFP251 deficiency caused glucose intolerance in mice. This phenotype was associated with impaired insulin signaling due to hypertrophic changes in white adipose tissue (WAT). Gene ontology analysis revealed that ZFP251 deficiency affected the expression of genes associated with adipocyte differentiation and lipid and fatty acid metabolism. Consistent with in vivo results, hypertrophic changes were observed in Zfp251 knockdown (KD) 3T3-L1 adipocytes. In addition, Zfp251 KD 3T3-L1 preadipocytes exhibited cell cycle arrest in G0/G1 phase, leading to impaired differentiation into mature adipocytes, upon which abnormal mitotic clonal expansion and reduced expression of adipogenic markers were exhibited. These results suggest that ZFP251 deficiency causes impaired adipogenesis and adipocyte hypertrophy, leading to dysfunction of WAT.

Mutation of Leaf Senescence 1 Encoding a C2H2 Zinc Finger Protein Induces ROS Accumulation and Accelerates Leaf Senescence in Rice.

Premature senescence of leaves causes a reduced yield and quality of rice by affecting plant growth and development. The regulatory mechanisms underlying early leaf senescence are still unclear. The Leaf senescence 1 (LS1) gene encodes a C2H2-type zinc finger protein that is localized to both the nucleus and cytoplasm. In this study, we constructed a rice mutant named leaf senescence 1 (ls1) with a premature leaf senescence phenotype using CRISPR/Cas9-mediated editing of the LS1 gene. The ls1 mutants exhibited premature leaf senescence and reduced chlorophyll content. The expression levels of LS1 were higher in mature or senescent leaves than that in young leaves. The contents of reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD) were significantly increased and catalase (CAT) activity was remarkably reduced in the ls1 plants. Furthermore, a faster decrease in pigment content was detected in mutants than that in WT upon induction of complete darkness. TUNEL and staining experiments indicated severe DNA degradation and programmed cell death in the ls1 mutants, which suggested that excessive ROS may lead to leaf senescence and cell death in ls1 plants. Additionally, an RT-qPCR analysis revealed that most senescence-associated and ROS-scavenging genes were upregulated in the ls1 mutants compared with the WT. Collectively, our findings revealed that LS1 might regulate leaf development and function, and that disruption of LS1 function promotes ROS accumulation and accelerates leaf senescence and cell death in rice.

Natural genetic variation in the HAIRS ABSENT (H) gene increases type-VI glandular trichomes in both wild and domesticated tomatoes

Glandular trichomes produce and exude secondary metabolites, conferring insect resistance in many crop species. Whereas some of its wild relatives are insect-resistant, tomato (Solanum lycopersicum) is not. Identifying the genetic changes that altered trichome development and biochemistry during tomato domestication would contribute to breeding for insect resistance. A mutation in the HAIRS ABSENT (H) gene, which encodes a C2H2 zinc finger protein (ZFP8), leads to reduced trichome density. Several geographic accessions of S. pimpinellifolium, the wild ancestor of domesticated tomato, have glabrous organs that resemble the phenotype caused by h. Here, we investigated allelic diversity for H in tomato and S. pimpinellifolium accessions and their associated trichome phenotypes. We also evaluated how the developmental stage can affect trichome development in glabrous and non-glabrous plants. We found that glabrous accessions of S. pimpinellifolium have different ZFP8 nucleotide sequence changes, associated with altered trichome development and density. We also found that while the glabrous appearance of h mutants is caused by a lower density of long trichomes, the density of type-VI glandular trichomes is increased, particularly in the adult stages of plant development. These insights on the genetic control of trichome development may contribute to breeding for insect resistance in tomatoes and other crops.

BaZFP1, a C2H2 Subfamily Gene in Desiccation-Tolerant Moss Bryum argenteum, Positively Regulates Growth and Development in Arabidopsis and Mosses.

C2H2 zinc finger protein (C2H2-ZFP) plays an important role in regulating plant growth, development, and response to abiotic stress. To date, there have been no analyses of the C2H2-ZFP family in desiccation-tolerant moss. In this study, we identified 57 BaZFP transcripts across the Bryum argenteum (B. argenteum) transcriptome. The BaZFP proteins were phylogenetically divided into four groups (I-IV). Additionally, we studied the BaZFP1 gene, which is a nuclear C2H2-ZFP and acts as a positive regulator of growth and development in both moss and Arabidopsis thaliana. The complete coding sequence of the BaZFP1 gene was isolated from B. argenteum cDNA, which showed a high expression level in a dehydration-rehydration treatment process. The overexpression of the BaZFP1 gene in the Physcomitrium patens and B. argenteum promoted differentiation and growth of gametophytes. Heterologous expression in Arabidopsis regulated the whole growth and development cycle. In addition, we quantitatively analyzed the genes related to growth and development in transgenic moss and Arabidopsis, including HLS1, HY5, ANT, LFY, FT, EIN3, MUS, APB4, SEC6, and STM1, and found that their expression levels changed significantly. This study may pave the way for substantial insights into the role of C2H2-ZFPs in plants as well as suggest appropriate candidate genes for crop breeding.

Genome-Wide Identification of C2H2 ZFPs and Functional Analysis of BRZAT12 under Low-Temperature Stress in Winter Rapeseed (Brassica rapa).

Zinc-finger protein (ZFP) transcription factors are among the largest families of transcription factors in plants. They participate in various biological processes such as apoptosis, autophagy, and stemness maintenance and play important roles in regulating plant growth and development and the response to stress. To elucidate the functions of ZFP genes in the low-temperature response of winter (Brassica rapa L.) B. rapa, this study identified 141 members of the C2H2 ZFP gene family from B. rapa, which are heterogeneously distributed on 10 chromosomes and have multiple cis-acting elements related to hormone regulation and abiotic stress of adversity. Most of the genes in this family contain only one CDS, and genes distributed in the same evolutionary branch share mostly the same motifs and are highly conserved in the evolution of cruciferous species. The genes were significantly upregulated in the roots and growth cones of ‘Longyou-7’, indicating that they play a role in the stress-response process of winter B. rapa. The expression level of the Bra002528 gene was higher in the strongly cold-resistant varieties than in the weakly cold-resistant varieties after low-temperature stress. The survival rate and BrZAT12 gene expression of trans-BrZAT12 Arabidopsis thaliana (Arabidopsis) were significantly higher than those of the wild-type plants at low temperature, and the enzyme activities in vivo were higher than those of the wild-type plants, indicating that the BrZAT12 gene could improve the cold resistance of winter B. rapa. BrZAT12 expression and superoxide dismutase and ascorbate peroxidase enzyme activities were upregulated in winter B. rapa after exogenous ABA treatment. BrZAT12 expression and enzyme activities decreased after the PD98059 treatment, and BrZAT12 expression and enzyme activities were higher than in the PD98059 treatment but lower than in the control after both treatments together. It is speculated that BrZAT12 plays a role in the ABA signaling process in which MAPKK is involved. This study provides a theoretical basis for the resolution of cold-resistance mechanisms in strong winter B. rapa.

The C2H2 zinc finger protein TaZFP13D increases drought stress tolerance in wheat

Wheat is one of the most cultivated crops, and its production is greatly affected by abiotic stresses such as drought. Transcription factors that regulate multiple downstream genes have a good potential to improve stress tolerance due to their ability to modulate various response pathways. Many members of the C1-2i C2H2 zinc finger protein (ZFP) sub-family are up-regulated in response to abiotic stress and specific members were shown to improve oxidative and abiotic stress tolerance in different plant species. Our previous work demonstrated that the most up-regulated TaZFP, TaZFP1B, improves drought tolerance and oxidative stress protection. Based on their strong response to different abiotic stresses, five additional candidate TaZFPs were selected for functional characterization by reverse genetics in three wheat lines, using the four-component Barley Stripe Mosaic Virus system. Of the five candidates studied, TaZFP13D was the only gene that increased drought stress tolerance (resistance to wilting and improved relative water content) in three different wheat lines and significantly improved antioxidant enzymes activities resulting in lower accumulation of superoxide and hydrogen peroxide. Moreover, TaZFP13D down-regulation decreased drought stress tolerance suggesting that this gene does not have a redundant function within the TaZFPs family.

The Drosophila ZAD zinc finger protein Kipferl guides Rhino to piRNA clusters.

RNA interference systems depend on the synthesis of small RNA precursors whose sequences define the target spectrum of these silencing pathways. The Drosophila Heterochromatin Protein 1 (HP1) variant Rhino permits transcription of PIWI-interacting RNA (piRNA) precursors within transposon-rich heterochromatic loci in germline cells. Current models propose that Rhino's specific chromatin occupancy at piRNA source loci is determined by histone marks and maternally inherited piRNAs, but also imply the existence of other, undiscovered specificity cues. Here, we identify a member of the diverse family of zinc finger associated domain (ZAD)-C2H2 zinc finger proteins, Kipferl, as critical Rhino cofactor in ovaries. By binding to guanosine-rich DNA motifs and interacting with the Rhino chromodomain, Kipferl recruits Rhino to specific loci and stabilizes it on chromatin. In kipferl mutant flies, Rhino is lost from most of its target chromatin loci and instead accumulates on pericentromeric Satellite arrays, resulting in decreased levels of transposon targeting piRNAs and impaired fertility. Our findings reveal that DNA sequence, in addition to the H3K9me3 mark, determines the identity of piRNA source loci and provide insight into how Rhino might be caught in the crossfire of genetic conflicts.

DEFECTIVELY ORGANIZED TRIBUTARIES 5 is not required for leaf venation patterning in Arabidopsis thaliana.

The search for genetic regulators of leaf venation patterning started over 30 years ago, primarily focused on mutant screens in the eudicotyledon Arabidopsis thaliana. Developmental perturbations in either cotyledons or true leaves led to the identification of transcription factors required to elaborate the characteristic reticulated vein network. An ortholog of one of these, the C2H2 zinc finger protein DEFECTIVELY ORGANIZED TRIBUTARIES 5 (AtDOT5), was recently identified through transcriptomics as a candidate regulator of parallel venation in maize (Zea mays) leaves. To elucidate how AtDOT5 regulates vein patterning, we generated three independent loss-of-function mutations by gene editing in Arabidopsis. Surprisingly, none of them exhibited any obvious phenotypic perturbations. To reconcile our findings with earlier reports, we re-evaluated the original Atdot5-1 and Atdot5-2 alleles. By genome sequencing, we show that reported mutations at the Atdot5-1 locus are actually polymorphisms between Landsberg erecta and Columbia ecotypes, and that other mutations present in the background most likely cause the pleiotropic mutant phenotype observed. We further show that a T-DNA insertion in the Atdot5-2 locus has no impact on leaf venation patterns when segregated from other T-DNA insertions present in the original line. We thus conclude that AtDOT5 plays no role in leaf venation patterning in Arabidopsis.

Epitope Fine Mapping by Mass Spectrometry: Investigations of Immune Complexes Consisting of Monoclonal Anti-HpTGEKP Antibody and Zinc Finger Protein Linker Phospho-Hexapeptides.

Accurate formation of antibody-antigen complexes has been relied on in both, multitudes of scientific projects and ample therapeutic and diagnostic applications. Mass spectrometrically determined dissociation behavior of immune complexes with the anti-HpTGEKP antibody revealed that the ten most frequently occurring phospho-hexapeptide linker sequences from C2H2 zinc finger proteins could be divided into two classes: orthodox binders, where strong noncovalent interactions developed as anticipated, and unorthodox binders with deviating structures and weaker binding. Phosphorylation of threonine was compulsory for antibody binding in an orthodox manner. Gas phase dissociation energy determinations of seven C2H2 zinc finger protein linker phospho-hexapeptides with orthodox binding properties revealed a bipolar binding motif of the antibody paratope. Epitope peptides, which in addition to the negatively charged phospho-threonine residue were C-terminally flanked by positively charged residues provided stronger binding, i. e. dissociation was endothermic, than peptides with acidic amino acid residues at these positions, for which dissociation was exothermic.