Zinc Finger-Homeodomain Transcriptional Factors (ZHDs) in Cucumber (Cucumis sativus L.): Identification, Evolution, Expression Profiles, and Function under Abiotic Stresses.

Cucumber (Cucumis sativus L.) is a globally prevalent and extensively cultivated vegetable whose yield is significantly influenced by various abiotic stresses, including drought, heat, and salinity. Transcription factors, such as zinc finger-homeodomain proteins (ZHDs), a plant-specific subgroup of Homeobox, play a crucial regulatory role in stress resistance. In this study, we identified 13 CsZHDs distributed across all six cucumber chromosomes except chromosome 7. Phylogenetic analysis classified these genes into five clades (ZHDI-IV and MIF) with different gene structures but similar conserved motifs. Collinearity analysis revealed that members of clades ZHD III, IV, and MIF experienced amplification through segmental duplication events. Additionally, a closer evolutionary relationship was observed between the ZHDs in Cucumis sativus (C. sativus) and Arabidopsis thaliana (A. thaliana) compared to Oryza sativa (O. sativa). Quantitative real-time PCR (qRT-PCR) analysis demonstrated the general expression of CsZHD genes across all tissues, with notable expression in leaf and flower buds. Moreover, most of the CsZHDs, particularly CsZHD9-11, exhibited varying responses to drought, heat, and salt stresses. Virus-induced gene silencing (VIGS) experiments highlighted the potential functions of CsZHD9 and CsZHD10, suggesting their positive regulation of stomatal movement and responsiveness to drought stress. In summary, these findings provide a valuable resource for future analysis of potential mechanisms underlying CsZHD genes in response to stresses.

In silico and computational analysis of zinc finger motif-associated homeodomain (ZF-HD) family genes in chilli (Capsicum annuum L).

Zinc finger-homeodomain (ZHD) proteins are mostly expressed in plants and are involved in proper growth and development and minimizing biotic and abiotic stress. A recent study identified and characterized the ZHD gene family in chilli (Capsicum annuum L.) to determine their probable molecular function. ZHD genes with various physicochemical characteristics were discovered on twelve chromosomes in chilli. We separated ZHD proteins into two major groups using sequence alignment and phylogenetic analysis. These groups differ in gene structure, motif distribution, and a conserved ZHD and micro-zinc finger ZF domain. The majority of the CaZHDs genes are preserved, early duplication occurred recently, and significant pure selection took place throughout evolution, according to evolutionary study. According to expression profiling, the genes were found to be equally expressed in tissues above the ground, contribute to plant growth and development and provide tolerance to biotic and abiotic stress. This in silico analysis, taken as a whole, hypothesized that these genes perform distinct roles in molecular and phytohormone signaling processes, which may serve as a foundation for subsequent research into the roles of these genes in other crops.

Enhancing the thermal stability and activity of zearalenone lactone hydrolase to promote zearalenone degradation via semi-rational design.

Zearalenone (ZEN) is a fungal toxin produced by Fusarium exospore, which poses a significant threat to both animal and human health due to its reproductive toxicity. Removing ZEN through ZEN lactonase is currently the most effective method reported, however, all published ZEN lactonases suffer from the poor thermal stability, losing almost all activity after 10 min of treatment at 55℃. In this study, we heterologously expressed ZHD11A from Phialophora macrospora and engineered it via semi-rational design. A mutant I160Y-G242S that can retain about 40 % residual activity at 55℃ for 10 min was obtained, which is the most heat-tolerant ZEN hydrolase reported to date. Moreover, the specific activity of the I160Y-G242S was also elevated 2-fold compared to ZHD11A from 220 U/mg to 450 U/mg, which is one of the most active ZEN lactonses reported. Dynamics analysis revealed that the decreased flexibility of the main-chain carbons contributes to increased thermal stability and the improved substrate binding affinity and catalytic turnover contribute to enhanced activity of variant I160Y-G242S. In all, the mutant I160Y-G242S is an excellent candidate for the industrial application of ZEN degradation.

Genome-Wide Identification and In Silico Analysis of ZF-HD Transcription Factor Genes in Zea mays L.

Zinc finger-homeodomain proteins are amongst the most prominent transcription factors (TFs) involved in biological processes, such as growth, development, and morphogenesis, and assist plants in alleviating the adverse effects of abiotic and biotic stresses. In the present study, genome-wide identification and expression analyses of the maize ZHD gene family were conducted. A total of 21 ZHD genes with different physicochemical properties were found distributed on nine chromosomes in maize. Through sequence alignment and phylogenetic analysis, we divided ZHD proteins into eight groups that have variations in gene structure, motif distribution, and a conserved ZF domain. Synteny analysis indicated duplication in four pairs of genes and the presence of orthologues of maize in monocots. Ka/Ks ratios suggested that strong pure selection occurred during evolution. Expression profiling revealed that the genes are evenly expressed in different tissues. Most of the genes were found to make a contribution to abiotic stress response, plant growth, and development. Overall, the evolutionary research on exons and introns, motif distributions, and cis-acting regions suggests that these genes play distinct roles in biological processes which may provide a basis for further study of these genes' functions in other crops.

Cloning and Characterization of Three Novel Enzymes Responsible for the Detoxification of Zearalenone.

Zearalenone is a common mycotoxin contaminant in cereals that causes severe economic losses and serious risks to health of human and animals. Many strategies have been devised to degrade ZEN and keep food safe. The hydrolase ZHD101 from Clonostachys rosea, which catalyzes the hydrolytic degradation of ZEN, has been studied widely. In the current research, three new enzymes that have the capacity to detoxify ZEN were identified, namely CLA, EXO, and TRI, showing 61%, 63%, and 97% amino acids identities with ZHD101, respectively. Three coding genes was expressed as heterologous in Escherichia coli BL21. Through biochemical analysis, the purified recombinant CLA, EXO, TRI, and ZHD101 exhibited high activities of degrading ZEN with the specific activity of 114.8 U/mg, 459.0 U/mg, 239.8 U/mg, and 242.8 U/mg. The optimal temperatures of CLA, EXO, TRI, and ZHD101 were 40 °C, 40 °C, 40 °C, and 45 °C, and their optimum pH were 7.0, 9.0, 9.5, and 9.0, respectively. Our study demonstrated that the novel enzymes CLA, EXO, and TRI possessed high ability to degrade ZEN from the model solutions and could be the promising candidates for ZEN detoxification in practical application.

Leaf rolling in bread wheat (Triticum aestivum L.) is controlled by the upregulation of a pair of closely linked/duplicate zinc finger homeodomain class transcription factors during moisture stress conditions.

Zinc finger-homeodomain (ZF-HDs) class IV transcriptional factors (TFs) is a plant-specific transcription factor and play a key role in stress responses, plant growth, development, and hormonal signaling. In this study, two new leaf rolling TFs genes, namely TaZHD1 and TaZHD10, were identified in wheat using comparative genomic analysis of the target region that carried a major QTL for leaf rolling identified through multi-environment phenotyping and high throughput genotyping of a RIL population. Structural and functional annotation of the candidate ZHD genes with its closest rice orthologs reflects the species-specific evolution and, undoubtedly, validates the notions of remote-distance homology concept. Meanwhile, the morphological analysis resulted in contrasting difference for leaf rolling in extreme RILs between parental lines HD2012 and NI5439 at booting and heading stages. Transcriptome-wide expression profiling revealed that TaZHD10 transcripts showed significantly higher expression levels than TaZHD1 in all leaf tissues upon drought stress. The relative expression of these genes was further validated by qRT-PCR analysis, which also showed consistent results across the studied genotypes at the booting and anthesis stage. The contrasting modulation of these genes under drought conditions and the available evidenced for its epigenetic behavior that might involve the regulation of metabolic and gene regulatory networks. Prediction of miRNAs resulted in five Tae-miRs that could be associated with RNAi mediated control of TaZHD1 and TaZHD10 putatively involved in the metabolic pathway controlling rolled leaf phenotype. Gene interaction network analysis indicated that TaZHD1 and TaZHD10 showed pleiotropic effects and might also involve other functions in wheat in addition to leaf rolling. Overall, the results increase our understanding of TaZHD genes and provide valuable information as robust candidate genes for future functional genomics research aiming for the breeding of wheat varieties tolerant to leaf rolling.

Zinc Finger-Homeodomain Genes: Evolution, Functional Differentiation, and Expression Profiling Under Flowering-Related Treatments and Abiotic Stresses in Plants.

Zinc finger-homeodomain (ZHD) proteins constitute a plant-specific transcription factor family that play important roles in plant growth, development, and stress responses. In this study, we investigated a total of 10, 17, and 31 ZHD gene members in the peach, Arabidopsis, and apple genome, respectively. The phylogenetic tree divided the identified ZHD genes into 4 subfamilies based on their domain organization, gene structure, and motif distribution with minor variations. The ZHD gene family members were unevenly distributed throughout in apple, peach, and Arabidopsis genomes. Segmental duplication was observed for 14 pairs of genes in apple. Transcript analysis found that ZHD genes mostly expressed in various tissues, particularly in leaves and flowers. Moreover, the transcript of most ZHD genes was significantly affected at different time points in response to various flowering-related exogenous hormones (sugar, gibberellin [GA], and 6-benzylaminopurine [6-BA]), signifying their possible role in the flowering induction in apple. Furthermore, the transcripts of CaZHD6, CaZHD7, CaZHD3, and CaZHD8 have induced in response to abiotic stresses including heat, drought, salt, and cold, indicating their possible involvement in response to abiotic stresses. Our research work systemically presents the different roles of ZHD genes. We believe that this study will provide a platform for future functional characterization of ZHD genes and to deeply unfold their roles in the regulation of flowering induction in plants.

Degrading Ochratoxin A and Zearalenone Mycotoxins Using a Multifunctional Recombinant Enzyme.

Zearalenone (ZEA) is an estrogenic and ochratoxin A (OTA) is a hepatotoxic Fusarium mycotoxin commonly seen in cereals and fruits products. No previous investigation has studied on a single platform for the multi degradation mycotoxin. The current study aimed to investigate the bifunctional activity of a novel fusion recombinant. We have generated a recombinant fusion enzyme (ZHDCP) by combining two single genes named zearalenone hydrolase (ZHD) and carboxypeptidase (CP) in frame deletion by crossover polymerase chain reaction (PCR). We identified enzymatic properties and cell cytotoxicity assay of ZHDCP enzyme. Our findings have demonstrated that ZEA was completely degraded to the non-toxic product in 2 h by ZHDCP enzyme at an optimum pH of 7 and a temperature of 35 °C. For the first time, it was found out that ZEA 60% was degraded by CP degrades in 48 h. Fusion ZHDCP and CP enzyme were able to degrade 100% OTA in 30 min at pH 7 and temperature 30 °C. ZEA- and OTA-induced cell death and increased cell apoptosis rate and regulated mRNA expression of Sirt1, Bax, Bcl2, Caspase3, TNFα, and IL6 genes. Our novel findings demonstrated that the fusion enzyme ZHDCP possess bifunctional activity (degrade OTA and ZEA), and it could be used to degrade more mycotoxins.

Zinc Finger-Homeodomain Transcriptional Factors (ZHDs) in Upland Cotton (Gossypium hirsutum): Genome-Wide Identification and Expression Analysis in Fiber Development.

Zinc finger-homeodomain (ZHD) genes encode a family of plant-specific transcription factors that not only participate in the regulation of plant growth and development but also play an important role in the response to abiotic stress. The ZHD gene family has been studied in several model plants, including Solanum lycopersicum, Zea mays, Oryza sativa, and Arabidopsis thaliana. However, a comprehensive study of the genes of the ZHD family and their roles in fiber development and pigmentation in upland cotton has not been completed. To address this gap, we selected a brown fiber cultivar for our study; brown color in cotton is one of the most desired colors in the textile industry. The natural colored fibers require less processing and little dying, thereby eliminating dye costs and chemical residues. Using bioinformatics approaches, we identified 37 GhZHD genes from Gossypium hirsutum and then divided these genes into seven groups based on their phylogeny. The GhZHD genes were mostly conserved in each subfamily with minor variations in motif distribution and gene structure. These genes were largely distributed on 19 of the 26 upland cotton chromosomes. Among the Gossypium genomes, the paralogs and orthologs of the GhZHD genes were identified and further characterized. Furthermore, among the paralogs, we observed that the ZHD family duplications in Gossypium genomes (G. hirsutum, G. arboreum, and G. raimondii) were probably derived from segmental duplication or genome-wide duplication (GWD) events. Through a combination of qRT-PCR and proanthocyanidins (PA) accumulation analyses in brown cotton fibers, we concluded that the candidate genes involved in early fiber development and fiber pigment synthesis include the following: GhZHD29, GhZHD35, GhZHD30, GhZHD31, GhZHD11, GhZHD27, GhZHD18, GhZHD15, GhZHD16, GhZHD22, GhZHD6, GhZHD33, GhZHD13, GhZHD5, and GhZHD23. This study delivers insights into the evolution of the GhZHD genes in brown cotton, serves as a valuable resource for further studies, and identifies the conditions necessary for improving the quality of brown cotton fiber.

Zearalenone detoxification by zearalenone hydrolase is important for the antagonistic ability of Clonostachys rosea against mycotoxigenic Fusarium graminearum.

The fungus Clonostachys rosea is antagonistic against plant pathogens, including Fusarium graminearum, which produces the oestrogenic mycotoxin zearalenone (ZEA). ZEA inhibits other fungi, and C. rosea can detoxify ZEA through the enzyme zearalenone lactonohydrolase (ZHD101). As the relevance of ZEA detoxification for biocontrol is unknown, we studied regulation and function of ZHD101 in C. rosea. Quantitative reverse-transcription PCR revealed zhd101 gene expression in all conditions studied and demonstrated dose-dependent induction by ZEA. Known inducers of the Polyketide Synthase pathway did not induce zhd101 expression, suggesting specificity of the enzyme towards ZEA. To assess the role of ZHD101 during biocontrol interactions, we generated two Δzhd101 mutants incapable of ZEA-detoxification and confirmed their defect in degrading ZEA by HPLC. The Δzhd101 mutants displayed a lower in vitro ability to inhibit growth of the ZEA-producing F. graminearum (strain 1104-14) compared to the wild type. In contrast, all three C. rosea strains equally inhibited growth of the F. graminearum mutant (ΔPKS4), which is impaired in ZEA-production. Furthermore, the Δzhd101 mutants failed to protect wheat seedlings against foot rot caused by the ZEA-producing F. graminearum. These data show that ZEA detoxification by ZHD101 is important for the biocontrol ability of C. rosea against F. graminearum.