Dof Proteins Research Articles

Genome-Based Identification of the Dof Gene Family in Three Cymbidium Species and Their Responses to Heat Stress in Cymbidium goeringii.

As an important genus in Orchidaceae, Cymbidium has rich ecological diversity and significant economic value. DNA binding with one zinc finger (Dof) proteins are pivotal plant-specific transcription factors that play crucial roles in the growth, development, and stress response of plants. Although the Dof genes have been identified and functionally analyzed in numerous plants, exploration in Orchidaceae remains limited. We conducted a thorough analysis of the Dof gene family in Cymbidium goeringii, C. ensifolium, and C. sinensis. In total, 91 Dof genes (27 CgDofs, 34 CeDofs, 30 CsDofs) were identified, and Dof genes were divided into five groups (I-V) based on phylogenetic analysis. All Dof proteins have motif 1 and motif 2 conserved domains and over half of the genes contained introns. Chromosomal localization and collinearity analysis of Dof genes revealed their evolutionary relationships and potential gene duplication events. Analysis of cis-elements in CgDofs, CeDofs, and CsDofs promoters showed that light-responsive cis-elements were the most common, followed by hormone-responsive elements, plant growth-related elements, and abiotic stress response elements. Dof proteins in three Cymbidium species primarily exhibit a random coil structure, while homology modeling exhibited significant similarity. In addition, RT-qPCR analysis showed that the expression levels of nine CgDofs changed greatly under heat stress. CgDof03, CgDof22, CgDof27, CgDof08, and CgDof23 showed varying degrees of upregulation. Most upregulated genes under heat stress belong to group I, indicating that the Dof genes in group I have great potential for high-temperature resistance. In conclusion, our study systematically demonstrated the molecular characteristics of Dof genes in different Cymbidium species, preliminarily revealed the patterns of heat stress, and provided a reference for further exploration of stress breeding in orchids.

DWARF AND LESS TILLERS ON CHROMOSOME 3 promotes tillering in rice by sustaining FLORAL ORGAN NUMBER 1 expression.

Three key factors determine yield in rice (Oryza sativa): panicle number, grain number, and grain weight. Panicle number is strongly associated with tiller number. Although many genes regulating tillering have been identified, whether Dof proteins are involved in controlling plant architecture remains unknown. The dwarf and less tillers on chromosome 3 (dlt3) rice mutant produces fewer tillers than the wild type. We cloned DLT3, which encodes a Dof protein that interacts with MONOCULM 3 (MOC3) in vivo and in vitro and recruits MOC1, forming a DLT3-MOC3-MOC1 complex. DLT3 binds to the promoter of FLORAL ORGAN NUMBER 1 (FON1) to activate its transcription and positively regulate tiller number. The overexpression of MOC1, MOC3, or FON1 in the dlt3 mutant increased tiller number. Collectively, these results suggest a model in which DLT3 regulates tiller number by maintaining the expression of MOC1, MOC3, and FON1. We discovered that DLT3 underwent directional selection in the Xian/indica and Geng/japonica populations during rice domestication. To provide genetic resources for breeding varieties with optimal panicle numbers, we performed large-scale diversity sequencing of the 1,080-bp DLT3 coding region of 531 accessions from different countries and regions. Haplotype analysis showed that the superior haplotype, DLT3H1, produced the most tillers, while haplotype DLT3H6 produced the fewest tillers. Our study provides important germplasm resources for breeding super high-yielding rice varieties with combinations of superior haplotypes in different target genes, which will help overcome the challenge of food and nutritional security in the future.

Genome-wide identification and molecular evolution of Dof transcription factors in Cyperus esculentus

Dof transcription factor family in Cyperus esculentus genome was identified and analyzed using bioinformatics. The analysis results revealed that C.esculentus genome contains 29 Dof genes (CesDof), all of which are located in the nucleus according to subcellular localization prediction. CesDof proteinrs have a range of 124 to 512 amino acids, with most being basic proteins. Their secondary structure was mainly irregular curl. The promoter sequence of CesDof genes contains cis-acting elements that respond to light, drought, hormones, low temperature, and circadian rhythm. Codon preference analysis showed that CesDof genes' codon preference ends in T/A. Collinearity analysis revealed that C.esculentus had three pairs of collinear CesDof genes. Additionally, there were 15 pairs of collinear genes between C.esculentus and Arabidopsis thaliana. The genetic relationship between C.esculentus and Rhynchospora pubera was found to be the closest. Phylogenetic tree analysis revealed that 29 CesDof genes of C.esculentus can be classified into 4 subgroups. Additionally, 144 miRNAs were predicted to target these CesDof genes. Furthermore, protein interaction analysis indicated that 15 Dof proteins in C.esculentus had interactions. The qRT-PCR verification results of drought stress and salt stress treatment experiments showed that most CesDof genes were involved in drought stress and salt stress responses, and the gene expression trends under drought stress and salt stress conditions were consistent. These results lay a theoretical foundation for further studying the molecular functions of Dof gene family in C.esculentus and its molecular mechanisms in regulating the life activities of C.esculentus.

DNA-Binding with One Finger (Dof) Transcription Factor Gene Family Study Reveals Differential Stress-Responsive Transcription Factors in Contrasting Drought Tolerance Potato Species.

DNA-binding with one finger (Dof) proteins comprise a large family that play central roles in stress tolerance by regulating the expression of stress-responsive genes via the DOFCORE element or by interacting with other regulatory proteins. Although the Dof TF has been identified in a variety of species, its systemic analysis in potato (Solanum tuberosum L.) is lacking and its potential role in abiotic stress responses remains unclear. A total of 36 potential Dof genes in potato were examined at the genomic and transcriptomic levels in this work. Five phylogenetic groups can be formed from these 36 Dof proteins. An analysis of cis-acting elements revealed the potential roles of Dofs in potato development, including under numerous abiotic stress conditions. The cycling Dof factors (CDFs) might be the initial step in the abiotic stress response signaling cascade. In potato, five CDFs (StCDF1/StDof19, StCDF2/StDof4, StCDF3/StDof11, StCDF4/StDof24, and StCDF5/StDof15) were identified, which are homologs of Arabidopsis CDFs. The results revealed that these genes were engaged in a variety of abiotic reactions. Moreover, an expression analysis of StDof genes in two potato cultivars ('Long10' (drought tolerant) and 'DXY' (drought susceptible)) of contrasting tolerances under drought stress was carried out. Further, a regulatory network mediated by lncRNA and its target Dofs was established. The present study provides fundamental knowledge for further investigation of the roles of Dofs in the adaptation of potato to drought stress, aiming to provide insights into a viable strategy for crop improvement and stress-resistance breeding.

Genome-wide identification, expression analysis and gene duplication analysis of Dof transcription factors between quinoa and its ancestral diploid sub-genome species reveal key CDF homologs involved in photoperiodic flowering regulation

Dof (DNA binding with One Finger) proteins are plant-specific transcription factors characteristic of a highly conserved DNA binding domain. Dof family members have been shown to play important roles in growth, development, stress defense, and light and phytohormone responses. In this study, we identified 38 CqDof genes in quinoa (Chenopodium quinoa). Phylogenetic analysis of Dof genes from quinoa, Arabidopsis, rice, sugar beet, C. pallidicaule and C. suecicum indicated that those Dofs were clustered into 6 sub-groups. Gene structure and conserved motif analysis showed that motif composition varied between different sub-groups. Duplication analysis indicated CqDof family was expanded mainly by segmental duplication, and we found the duplication events CqDof31/CqDof36 and CqDof31/CqDof37 could be traced back to before the divergence of Chenopodium ancestor from sugar beet, and a recent duplication event CqDof33/CqDof34 was detected specific to quinoa. Analysis of cis-acting elements showed that CqDof genes may be involved in light, phytohormone and stress response. Expression pattern analysis showed that CqDof27, CqDof31/CqDof32, CqDof33/CqDof34 and CqDof35/CqDof36 in sub-group VI were affected by different photoperiods and they may act as important floral regulator. Based on the cis-element analysis and diurnal expression results, we speculated the recent duplication event CqDof33/CqDof34 may render quinoa some growth habits to adapt to different latitudes, and CqDof31/CqDof32 might be potential candidates for inflorescence branching and yield improvement. Together, the results obtained in this study will help understand the functions of Dof genes in quinoa.

Genome-wide identification and functional analysis of Dof transcription factor family in Camelina sativa

BackgroundDof transcription factors (TFs) containing C2-C2 zinc finger domains are plant-specific regulatory proteins, playing crucial roles in a variety of biological processes. However, little is known about Dof in Camelina sativa, an important oil crop worldwide, with high stress tolerance. In this study, a genome-wide characterization of Dof proteins is performed to examine their basic structural characteristics, phylogenetics, expression patterns, and functions to identify the regulatory mechanism underlying lipid/oil accumulation and the candidate Dofs mediating stress resistance regulation in C. sativa.ResultsTotal of 103 CsDof genes unevenly distributed on 20 chromosomes were identified from the C. sativa genome, and they were classified into four groups (A, B, C and D) based on the classification of Arabidopsis Dof gene family. All of the CsDof proteins contained the highly-conserved typic CX2C-X21-CX2C structure. Segmental duplication and purifying selection were detected for CsDof genes. 61 CsDof genes were expressed in multiple tissues, and 20 of them showed tissue-specific expression patterns, suggesting that CsDof genes functioned differentially in different tissues of C. sativa. Remarkably, a set of CsDof members were detected to be possible involved in regulation of oil/lipid biosynthesis in C. sativa. Six CsDof genes exhibited significant expression changes in seedlings under salt stress treatment.ConclusionsThe present data reveals that segmental duplication is the key force responsible for the expansion of CsDof gene family, and a strong purifying pressure plays a crucial role in CsDofs’ evolution. Several CsDof TFs may mediate lipid metabolism and stress responses in C. sativa. Several CsDof TFs may mediate lipid metabolism and stress responses in C. sativa. Collectively, our findings provide a foundation for deep understanding the roles of CsDofs and genetic improvements of oil yield and salt stress tolerance in this species and the related crops.

Comprehensive in silico analysis of Phospholipase D gene family in economically important orchids

• A total of 25 putative PLD proteins from the genomes of Phalaenopsis equestris (7) , Dendrobium catenatum (9) and Apostasia shenzhenica (9) were identified. • These proteins are comprised of two HKD domains and two additional domains; C2 and PXPH domains at N-terminal region. • The multiple sequence alignment, domain and motifs analysis revealed their conserved nature and homology structure simulation and Ramachadran plot analysis revealed the abundance of beta sheets in tertiary structures. • The presence of Root-specific element, DOF proteins binding domain, scaffold or matrix attachment region, pollen-specific, dehydration-responsive, light-responsive, gibberellin-responsive, ABA-responsive elements and Meja-responsive upstream cis- regulatory promoter elements indicated their role in various physiological processes. Phospholipase D (PLD) plays a fundamental role in various aspects of plant growth, development and stress responses. In present study, 25 putative PLD proteins from the genomes of Phalaenopsis equestris (7) , Dendrobium catenatum (9) and Apostasia shenzhenica (9) were identified on the basis of presence of two HKD domains and two additional domains; C2 and PXPH domains at N-terminal region. These proteins were clustered into five sub-groups (α, β, δ, ζ and φ) along with their orthologs in Arabidopsis thaliana and Oryza sativa upon phylogenetic analysis. Gene characterization divulged that most of the PePLD, DcPLD and AsPLD genes within the same sub-group exhibited similar exon-intron organization and highly conserved motifs. RNA-seq based spatio-temporal studies indicated differential expression in various vegetative and reproductive tissues. PePLDα 1 , DcPLDα 1 and AsPLDα 1 were showing significant expression in all the tissues, while for other genes the expression was limited either in vegetative or reproductive tissues. Homology structure simulation revealed the abundance of random coils in secondary structures and beta sheets in tertiary structures. The presence of specific upstream cis- regulatory promoter elements emphasized the role of PLD s in various physiological processes. This report is the first genome-wide characterization of the PLD gene family in orchids and shall provide new insight into the functional characterization of these genes.

Genome-Wide Identification of DOF Gene Family and the Mechanism Dissection of SbDof21 Regulating Starch Biosynthesis in Sorghum.

Starch is one of the main utilization products of sorghum (Sorghum bicolor L.), the fifth largest cereal crop in the world. Up to now, the regulation mechanism of starch biosynthesis is rarely documented in sorghum. In the present study, we identified 30 genes encoding the C2-C2 zinc finger domain (DOF), with one to three exons in the sorghum genome. The DOF proteins of sorghum were divided into two types according to the results of sequence alignment and evolutionary analysis. Based on gene expressions and co-expression analysis, we identified a regulatory factor, SbDof21, that was located on chromosome 5. SbDof21 contained two exons, encoding a 36.122 kD protein composed of 340 amino acids. SbDof21 co-expressed with 15 genes involved in the sorghum starch biosynthesis pathway, and the Pearson correlation coefficients (PCCs) with 11 genes were greater than 0.9. The results of qRT-PCR assays indicated that SbDof21 is highly expressed in sorghum grains, exhibiting low relative expression levels in the tissues of roots, stems and leaves. SbDOF21 presented as a typical DOF transcription factor (TF) that was localized to the nucleus and possessed transcriptional activation activity. Amino acids at positions 182–231 of SbDOF21 formed an important structure in its activation domain. The results of EMSA showed that SbDOF21 could bind to four tandem repeats of P-Box (TGTAAAG) motifs in vitro, such as its homologous proteins of ZmDOF36, OsPBF and TaPBF. Meanwhile, we also discovered that SbDOF21 could bind and transactivate SbGBSSI, a key gene in sorghum amylose biosynthesis. Collectively, the results of the present study suggest that SbDOF21 acts as an important regulator in sorghum starch biosynthesis, exhibiting potential values for the improvement of starch contents in sorghum.

Genome-wide identification, phylogenetic and expression pattern analysis of Dof transcription factors in blueberry (Vaccinium corymbosum L.).

BackgroundDNA binding with one finger (Dof) proteins are plant-specific transcription factor (TF) that plays a significant role in various biological processes such as plant growth and development, hormone regulation, and resistance to abiotic stress. The Dof genes have been identified and reported in multiple plants, but so far, the whole genome identification and analysis of Dof transcription factors in blueberry (Vaccinium corymbosum L.) have not been reported yet.MethodsUsing the Vaccinium genome, we have identified 51 VcDof genes in blueberry. We have further analyzed their physicochemical properties, phylogenetic relationships, gene structure, collinear analysis, selective evolutionary pressure, cis-acting promoter elements, and tissue and abiotic stress expression patterns.ResultsFifty-one VcDof genes were divided into eight subfamilies, and the genes in each subfamily contained similar gene structure and motif ordering. A total of 24 pairs of colinear genes were screened; VcDof genes expanded mainly due to whole-genome duplication, which was subjected to strong purifying selection pressure during the evolution. The promoter of VcDof genes contains three types of cis-acting elements for plant growth and development, phytohormone and stress defense responsiveness. Expression profiles of VcDof genes in different tissues and fruit developmental stages of blueberry indicated that VcDof2 and VcDof45 might play a specific role in anthesis and fruit growth and development. Expression profiles of VcDof genes in different stress indicated that VcDof1, VcDof11, and VcDof15 were highly sensitive to abiotic stress. This study provides a theoretical basis for further clarifying the biological function of Dof genes in blueberry.

Emerging Roles of Plant DNA-Binding With One Finger Transcription Factors in Various Hormone and Stress Signaling Pathways.

Coordinated transcriptional regulation of stress-responsive genes orchestrated by a complex network of transcription factors (TFs) and the reprogramming of metabolism ensure a plant’s continued growth and survival under adverse environmental conditions (e.g., abiotic stress). DNA-binding with one finger (Dof) proteins, a group of plant-specific TF, were identified as one of several key components of the transcriptional regulatory network involved in abiotic stress responses. In many plant species, Dofs are often activated in response to a wide range of adverse environmental conditions. Dofs play central roles in stress tolerance by regulating the expression of stress-responsive genes via the DOFCORE element or by interacting with other regulatory proteins. Moreover, Dofs act as a key regulatory hub of several phytohormone pathways, integrating abscisic acid, jasmonate, SA and redox signaling in response to many abiotic stresses. Taken together, we highlight a unique role of Dofs in hormone and stress signaling that integrates plant response to adverse environmental conditions with different aspects of plant growth and development.

Genome-Wide Identification of DNA Binding with One Finger (Dof) Gene Family in Tartary Buckwheat (Fagopyrum tataricum) and Analysis of Its Expression Pattern after Exogenous Hormone Stimulation.

Simple SummaryA number of studies have demonstrated that DNA binding with one finger (Dof) proteins are involved in multiple biological processes. In the present study, Dof genes or proteins in Tartary buckwheat (FtDofs) were systematically analysed, including their physical properties, phylogenetic relationships, structure, motif composition, cis-acting elements present in promoter regions, chromosomal distribution, gene duplication events, syntenic relationships, expression patterns in different tissues and different fruit developmental stages and responses to exogenous hormone stimulation. The results indicated that the expansion of FtDofs was mainly due to segmental duplication. The tissue-specific expression patterns of FtDofs and their positive responses to exogenous hormone stimulation suggest that they play important roles in the growth and development of Tartary buckwheat as well as in the adaptation to environmental changes. Collectively, this study lays a foundation for further exploration of the function of FtDof genes in Tartary buckwheat.DNA binding with one finger (Dof) proteins have been proven to be involved in multiple biological processes. However, genome-wide identification of the Dof gene family has not been reported for Tartary buckwheat (Fagopyrum tataricum). In this study, 35 FtDof proteins were identified, and they could be divided into nine phylogenetic subgroups. Proteins within the same subgroup had similar gene structure and motif composition. Moreover, abundant cis-acting elements were present in the promoter regions of FtDof genes. Segmental duplication was the primary driving force for the evolution of the FtDof gene family. Synteny analysis indicated that Tartary buckwheat was closer to dicotyledons, and more orthologous Dof genes existed among them. The expression pattern of FtDofs in different tissues and at different fruit developmental stages varied. Different tissues contained several genes that were specifically expressed. FtDof expression was mainly upregulated under methyl jasmonate treatment and downregulated under other hormone treatments. Taken together, FtDofs may play important roles in the growth and development of Tartary buckwheat and in response to abiotic and biotic stresses. Therefore, the genome-wide identification and expression pattern analysis of the Tartary buckwheat Dof gene family lays a foundation for further exploration of the functional characteristics of FtDofs in the future.

Genome-wide analysis of Dof transcription factors and their response to cold stress in rice (Oryza sativa L.)

BackgroundRice (Oryza sativa L.) is a food crop for humans worldwide. However, temperature has an effect during the vegetative and reproductive stages. In high-latitude regions where rice is cultivated, cold stress is a major cause of yield loss and plant death. Research has identified a group of plant-specific transcription factors, DNA binding with one zinc fingers (DOFs), with a diverse range of functions, including stress signaling and stress response during plant growth. The aim of this study was to identify Dof genes in two rice subspecies, indica and japonica, and screen for Dof genes that may be involved in cold tolerance during plant growth.ResultsA total of 30 rice Dofs (OsDofs) were identified using bioinformatics and genome-wide analyses and phylogenetically analyzed. The 30 OsDOFs were classified into six subfamilies, and 24 motifs were identified based on protein sequence alignment. The chromosome locations of OsDofs were determined and nine gene duplication events were identified. A joint phylogenetic analysis was performed on DOF protein sequences obtained from four monocotyledon species to examine the evolutionary relationship of DOF proteins. Expression profiling of OsDofs from two japonica cultivars (Longdao5, which is cold-tolerant, and Longjing11, which is cold-sensitive) revealed that OsDof1 and OsDof19 are cold-inducible genes. We examined the seed setting rates in OsDof1- and OsDof19-overexpression and RNAi lines and found that OsDof1 showed a response to cold stress.ConclusionsOur investigation identified OsDof1 as a potential target for genetic breeding of rice with enhanced cold tolerance.

Genome-wide identification of the DNA-binding one zinc finger (Dof) transcription factor gene family and their potential functioning in nitrogen remobilization in tea plant (Camellia sinensis L.)

The DNA-binding one zinc finger (Dof) proteins are a type of transcription factor (TF) specific to plants that are involved in various aspects of their biological processes. Although the Dof TF has been identified in a variety of species, its systemic analysis in tea (Camellia sinensis L.) is lacking and its potential role in this plant's nitrogen remobilization remains unclear. Here, we carried out a genome-wide identification of Dofs in tea and identified 41 CsDofs at the chromosome level. Their genetic structure, conserved motif, and synteny relations were analyzed and visualized, using information extracted from a newly available genome database. Phylogenetic analysis of Dofs from seven different plant species revealed that Dof members could be clustered into six groups. Heatmap analysis showed that CsDofs have similar expression patterns among different species of genus Camellia, and tissue-specific transcription patterns among tea tissues. Expression patterns of CsDofs in mature leaves during the development of new shoots revealed that most of CsDofs peaked on March 24 when the new shoots developed into one bud and two leaves. Furthermore, the expression pattern of CsDof13/16/29/33/34/35/36/39 was significantly correlated with that of genes involved in N metabolism, such as GDH or GS, indicating these CsDofs might play roles in regulating the expression of genes participating in N metabolism of tea. Taken together, this study provides information on the genome-wide identification of the CsDofs’ family in tea, and identifies putative candidates possible involved in N remobilization in mature leaves during the development of new shoots in spring.

Genome-wide binding analysis of the tomato transcription factor SlDof1 reveals its regulatory impacts on fruit ripening

The DNA binding with one finger (Dof) proteins are plant-specific transcription factors involved in a variety of biological processes. However, little is known about their functions in fruit ripening, a flowering-plant-specific process that is required for seed maturation and dispersal. Here, we found that the tomato Dof transcription factor SlDof1, is necessary for normal fruit ripening. Knockdown of SlDof1 expression by RNA interference delayed ripening-related processes, including lycopene synthesis and ethylene production. Transcriptome profiling indicated that SlDof1 influences the expression of hundreds of genes, and a chromatin immunoprecipitation sequencing revealed a large number of SlDof1 binding sites. A total of 312 genes were identified as direct targets of SlDof1, among which 162 were negatively regulated by SlDof1 and 150 were positively regulated. The SlDof1 target genes were involved in a variety of metabolic pathways, and follow-up analyses verified that SlDof1 directly regulates some well-known ripening-related genes including ACS2 and PG2A as well as transcriptional repressor genes such as SlIAA27. Our findings provide insights into the transcriptional regulatory networks underlying fruit ripening and highlight a gene potentially useful for genetic engineering to control ripening.

Genome-wide analysis of BpDof genes and the tolerance to drought stress in birch (Betula platyphylla).

BackgroundDNA binding with one finger (Dof) proteins are plant-specific transcription factors playing vital roles in developmental processes and stress responses in plants. Nevertheless, the characterizations, expression patterns, and functions of the Dof family under drought stress (a key determinant of plant physiology and metabolic homeostasis) in woody plants remain unclear.MethodsThe birch (Betula platyphylla var. mandshuric) genome and plant TFDB database were used to identify Dof gene family members in birch plants. ClustalW2 of BioEdit v7.2.1, MEGA v7.0, ExPASy ProtParam tool, Subloc, TMHMM v2.0, GSDS v2.0, MEME, TBtools, KaKs Calculator v2.0, and PlantCARE were respectively used to align the BpDof sequences, build a phylogenetic tree, identify the physicochemical properties, analyze the chromosomal distribution and synteny, and identify the cis-elements in the promoter regions of the 26 BpDof genes. Additionally, the birch seedlings were exposed to PEG6000-simulated drought stress, and the expression patterns of the BpDof genes in different tissues were analyzed by qRT-PCR. The histochemical staining and the evaluation of physiological indexes were performed to assess the plant tolerance to drought with transient overexpression of BpDof4, BpDof11, and BpDof17 genes. SPSS software and ANOVA were used to conduct all statistical analyses and determine statistically significant differences between results.ResultsA total of 26 BpDof genes were identified in birch via whole-genome analysis. The conserved Dof domain with a C(x)2C(x)21C(x)2C zinc finger motif was present in all BpDof proteins. These birch BpDofs were classified into four groups (A to D) according to the phylogenetic analysis of Arabidopsis thaliana Dof genes. BpDof proteins within the same group mostly possessed similar motifs, as detected by conserved motif analysis. The exon–intron analysis revealed that the structures of BpDof genes differed, indicating probable gene gain and lose during the BpDof evolution. The chromosomal distribution and synteny analysis showed that the 26 BpDofs were unevenly distributed on 14 chromosomes, and seven duplication events among six chromosomes were found. Cis-acting elements were abundant in the promoter regions of the 26 BpDof genes. qRT-PCR revealed that the expression of the 26 BpDof genes was differentially regulated by drought stress among roots, stems, and leaves. Most BpDof genes responded to drought stress, and BpDof4, BpDof11, and BpDof17­ were significantly up-regulated. Therefore, plants overexpressing these three genes were generated to investigate drought stress tolerance. The BpDof4-, BpDof11-, and BpDof17­-overexpressing plants showed promoted reactive oxygen species (ROS) scavenging capabilities and less severe cell damage, suggesting that they conferred enhanced drought tolerance in birch. This study provided an in-depth insight into the structure, evolution, expression, and function of the Dof gene family in plants.

Genome-wide analysis and functional characterization of the Dof transcription factor family in rice (Oryza sativa L.)

Exhaustive searches of the rice genome have revealed 30 different potential OsDof (Oryza sativa DNA binding with One Finger) genes. Their subcellular localization, phylogenetic relationship, conserved motifs identification, chromosomal allocation, expression patterns, and interaction networks were analyzed. The Dof (DNA binding with One Finger) family of transcription factors represents a particular class of plant-specific transcriptional regulators, contain a highly conserved region of 50-52 amino acids (Dof domain) and involved in various plant developmental processes and response to various environmental stresses. Few (Oryza sativa) OsDof genes have been demonstrated previously for their biological functions but there is no comprehensive study on most of the Dof genes of rice. In the current study, exhaustive searches of the rice genome revealed 30 different potential OsDof genes, and then their subcellular localization, phylogenetic relationship, conserved motifs identification, chromosomal allocation, expression patterns, and interaction networks were analyzed. Phylogenetic analysis of Dof proteins in rice showed that they are distributed in 4 groups. By genome-wide observation of gene expression profiles, we found that OsDof genes showed significant variances in expression levels in different tissues across multiple developmental stages. Protein-protein correlation network analysis, shows a statically significant overlap of some OsDofs, suggesting their similar functions and a high degree of co-expression. The Dof family transcription factors have been reported for their involvement in the regulation of various gene expression processes in rice but still, most of the Dof genes are not characterized for their specific physiological functions. This study revealed useful information and clues about predicting the potential roles of OsDofs in rice by combining their genome-wide characterization, expression profiling, protein-protein interactions, and for further studies to develop high-quality rice varieties.

Genome-Wide Analysis of Dof Genes and Their Response to Abiotic Stress in Rose (Rosa chinensis).

Dof (DNA binding with one finger) proteins play important roles in plant development and defense regulatory networks. In the present study, we report a genome-wide analysis of rose Dof genes (RchDof), including phylogenetic inferences, gene structures, chromosomal locations, gene duplications, and expression diversity. A total of 24 full-length RchDof genes were identified in Rosa chinensis, which were assigned to nine distinct subgroups. These RchDof genes were unevenly distributed on rose chromosomes. The genome-scale analysis of synteny indicated that segmental duplication events may have played a major role in the evolution of the RchDof gene family. Analysis of cis-acting elements revealed putative functions of Dofs in rose during development as well as under numerous biotic and abiotic stress conditions. Moreover, the expression profiles derived from qRT-PCR experiments demonstrated distinct expression patterns in various tissues, and gene expression divergence existed among the duplicated RchDof genes, suggesting a fundamentally functional divergence of the duplicated Dof paralogs in rose. The gene expression analysis of RchDofs under drought and salt stress conditions was also performed. The present study offered novel insights into the evolution of RchDofs and can aid in the further functional characterization of its candidate genes.

Transcriptomic analysis of grapevine Dof transcription factor gene family in response to cold stress and functional analyses of the VaDof17d gene

Dof genes enhance cold tolerance in grapevine and VaDof17d is tightly associated with the cold-responsive pathway and with the raffinose family oligosaccharides. DNA-binding with one finger (Dof) proteins comprise a large family that plays important roles in the regulation of abiotic stresses. No in-depth analysis of Dof genes has been performed in the grapevine. In this study, we analyzed a total of 25 putative Dof genes in grapevine at genomic and transcriptomic levels, compiled expression profiles of 11 selected VaDof genes under cold stress and studied the potential function of the VaDof17d gene in grapevine calli. The 25 Dof proteins can be classified into four phylogenetic groups. RNA-seq and qRT-PCR results demonstrated that a total of 11 VaDof genes responded to cold stress. Comparative mRNA sequencing of 35S::VaDof17d grape calli showed that VaDof17d was tightly associated with the cold-responsive pathway and with the raffinose family oligosaccharides (RFOs), as observed by the up-regulation of galactinol synthase (GolS) and raffinose synthase genes. We found that the Dof17d-ED (CRISPR/Cas9-mediated mutagenesis of Dof17d-ED) mutant had low cold tolerance with a decreased RFOs level during cold stress. These results formed the fundamental knowledge for further analysis of the biological roles of Dof genes in the grapevine's adaption to cold stresses.

Identification and characterization of SlbHLH, SlDof and SlWRKY transcription factors interacting with SlDPD gene involved in costunolide biosynthesis in Saussurea lappa

The genes involved in costunolide biosynthesis in Saussurea lappa have been identified recently by our lab. However, the study of transcriptional regulators of these genes was lacking for better opportunities for engineering the pharmacologically important biosynthetic pathway. Therefore, we cloned the promoter region of diphosphomevalonate decarboxylase gene (DPD) and analyzed its cis-acting regulatory elements to reveal the potential transcription factor (TF) binding sites for Dof, bHLH and WRKY family proteins in the gene promoter. The transcriptome study approach followed by the hidden Markov model based search, digital gene expression, co-expression network analysis, conserved domain properties and evolutionary analyses were carried out to screen out seven putative TFs for the DPD-TF interaction studies. Yeast one-hybrid assays were performed and three TFs were reported, namely, SlDOF2, SlbHLH3 and SlWRKY2 from Dof, bHLH and WRKY families, respectively that interacted positively with the DPD gene of the costunolide biosynthetic pathway. The tissue specific relative gene expression studies also supported the linked co-expression of the gene and its interacting TFs The present report will improve the understanding of transcriptional regulation pattern of costunolide biosynthetic pathway.

The apple DNA-binding one zinc-finger protein MdDof54 promotes drought resistance

DNA-binding one zinc-finger (Dof) proteins constitute a family of transcription factors with a highly conserved Dof domain that contains a C2C2 zinc-finger motif. Although several studies have demonstrated that Dof proteins are involved in multiple plant processes, including development and stress resistance, the functions of these proteins in drought stress resistance are largely unknown. Here, we report the identification of the MdDof54 gene from apple and document its positive roles in apple drought resistance. After long-term drought stress, compared with nontransgenic plants, MdDof54 RNAi plants had significantly shorter heights and weaker root systems; the transgenic plants also had lower shoot and root hydraulic conductivity, as well as lower photosynthesis rates. By contrast, compared with nontransgenic plants, MdDof54-overexpressing plants had higher photosynthesis rates and shoot hydraulic conductivity under long-term drought stress. Moreover, compared with nontransgenic plants, MdDof54-overexpressing plants had higher survival percentages under short-term drought stress, whereas MdDof54 RNAi plants had lower survival percentages. MdDof54 RNAi plants showed significant downregulation of 99 genes and significant upregulation of 992 genes in response to drought, and 366 of these genes were responsive to drought. We used DAP-seq and ChIP-seq analyses to demonstrate that MdDof54 recognizes cis-elements that contain an AAAG motif. Taken together, our results provide new information on the functions of MdDof54 in plant drought stress resistance as well as resources for apple breeding aimed at the improvement of drought resistance.