Plant Transcription Factors Research Articles

Anthocyanin Biosynthesis Induced by MYB Transcription Factors in Plants.

Anthocyanins act as polyphenolic pigment that is ubiquitously found in plants. Anthocyanins play a role not only in health-promoting as an antioxidant, but also in protection against all kinds of abiotic and biotic stresses. Most recent studies have found that MYB transcription factors (MYB TFs) could positively or negatively regulate anthocyanin biosynthesis. Understanding the roles of MYB TFs is essential in elucidating how MYB TFs regulate the accumulation of anthocyanin. In the review, we summarized the signaling pathways medicated by MYB TFs during anthocyanin biosynthesis including jasmonic acid (JA) signaling pathway, cytokinins (CKs) signaling pathway, temperature-induced, light signal, 26S proteasome pathway, NAC TFs, and bHLH TFs. Moreover, structural and regulator genes induced by MYB TFs, target genes bound and activated or suppressed by MYB TFs, and crosstalk between MYB TFs and other proteins, were found to be vitally important in the regulation of anthocyanin biosynthesis. In this study, we focus on the recent knowledge concerning the regulator signaling and mechanism of MYB TFs on anthocyanin biosynthesis, covering the signaling pathway, genes expression, and target genes and protein expression.

Plastidic G6PDH and root structure regulation are essential for high nitrogen use efficiency in highland barley adaptation to low nitrogen

Nitrogen (N) use efficiency (NUE) is a major factor determining crop growth and productivity. It is closely related to N acquisition and assimilation efficiency. In this study, the respective responses and mechanisms of highland barley (Kunlun14) and barley (Ganpi6) were investigated under low N condition. The root length, plant height, root/shoot ratio, and relative root growth rate in Kunlun14 were greater than those in Ganpi6 under low N stress, suggesting Kunlun14 has stronger tolerance to low N. The contents of nitrate and amino acids were decreased more in Ganpi6 than in Kunlun14 under low N stress. Moreover, Kunlun14 maintained higher nitrate uptake, translocation and assimilation capacity than Ganpi6. Glucose-6-phosphate dehydrogenase (G6PDH) activity, especially the plastidic G6PDH (Pla-G6PDH) activity, was markedly induced by low N in Kunlun14. Glucosamine, a G6PDH inhibitor, reduced nitrate uptake and assimilation capacity under low N stress. qRT-PCR and RNA-Seq analysis showed that the expression of HvG6PDH3 , encoding Pla-G6PDH3, was markedly induced by low N in barley roots. In addition, the differentially expressed genes in barley roots involved in hormones, transcription factors and cell wall biosynthesis play key roles under low N by regulating root development and N metabolism. Overexpression of HvG6PDH3 in Arabidopsis up-regulated the expression of AtNRT1.5 and AtNRT2.1 and increased the nitrate uptake and translocation capacity, which resulted in higher seed yield in HvG6PDH3 -overexpressors under low N stress. Taken together, HvG6PDH3 and the genes involved in regulating root architecture play essential roles in Kunlun14 tolerance to low N, which is conductive to maintaining high N uptake and assimilation efficiency. • Highland barley (Kunlun14) has higher NUE than barley (Ganpi6) under low N stress. • HvG6PDH3 is involved in the tolerance of highland barley to low N by positively regulating N uptake and assimilation. • DEGs involved in plant hormones, transcription factors and cell wall biosynthesis play key roles under low N stress.

Genome-wide identification and expression analysis of WRKY family genes under soft rot in Chinese cabbage.

Complex transcriptional networks regulate plant defense against pathogen attack, and plant transcription factors act as key regulators of the plant immune responses. The differences between transcription factor expression and regulation in Chinese cabbage soft rot (Pectobacterium carotovorum; Pc) have not been revealed. In this study, a total of 148 putative Chinese cabbage WRKY genes (BrWRKYs) were identified from the Chinese cabbage genome (v3.0). These genes were divided into seven subgroups (groups I, IIa–e, and III) based on phylogenomic analysis, with distinct motif compositions in each subgroup. Time-series RNA-seq was carried out to elucidate the dynamic expression patterns of the BrWRKYs on the resistant mutant (sr) and the susceptible wild-type (inbred WT) challenged by Pc. Transcriptional analysis showed that 48 WRKY transcription genes at 0–24 hpi were significantly upregulated in sr under soft rot stress. At the 12-h post-inoculation critical time point, we identified three specifically upregulated genes and two downregulated genes in the resistant mutant, which may provide potential applications for genetic improvement against soft rot. The findings improved our understanding of the WRKY-mediated soft rot stress response regulation in Chinese cabbage. The study thus lays a foundation for the genetic improvement of soft rot resistance.

PlantBind: an attention-based multi-label neural network for predicting plant transcription factor binding sites.

Identification of transcription factor binding sites (TFBSs) is essential to understanding of gene regulation. Designing computational models for accurate prediction of TFBSs is crucial because it is not feasible to experimentally assay all transcription factors (TFs) in all sequenced eukaryotic genomes. Although many methods have been proposed for the identification of TFBSs in humans, methods designed for plants are comparatively underdeveloped. Here, we present PlantBind, a method for integrated prediction and interpretation of TFBSs based on DNA sequences and DNA shape profiles. Built on an attention-based multi-label deep learning framework, PlantBind not only simultaneously predicts the potential binding sites of 315 TFs, but also identifies the motifs bound by transcription factors. During the training process, this model revealed a strong similarity among TF family members with respect to target binding sequences. Trans-species prediction performance using four Zea mays TFs demonstrated the suitability of this model for transfer learning. Overall, this study provides an effective solution for identifying plant TFBSs, which will promote greater understanding of transcriptional regulatory mechanisms in plants.

Genome-wide analysis and molecular dissection of the SPL gene family in Fraxinus mandshurica

BackgroundSQUAMOSA promoter binding protein-like (SPL) is a unique family of transcription factors in plants, which is engaged in regulating plant growth and development, physiological and biochemical processes. Fraxinus mandshurica is an excellent timber species with a wide range of uses in northeastern China and enjoys a high reputation in the international market. SPL family analysis has been reported in some plants while SPL family analysis of Fraxinus mandshurica has not been reported.ResultsWe used phylogeny, conserved motifs, gene structure, secondary structure prediction, miR156 binding sites, promoter cis elements and GO annotation to systematically analyze the FmSPLs family. This was followed by expression analysis by subcellular localization, expression patterns at various tissue sites, abiotic stress and hormone induction. Because FmSPL2 is highly expressed in flowers it was selected to describe the SPL gene family of Fraxinus mandshurica by ectopic expression. Among them, 10 FmSPL genes that were highly expressed at different loci were selected for expression analysis under abiotic stress (NaCl and Cold) and hormone induction (IAA and ABA). These 10 FmSPL genes showed corresponding trends in response to both abiotic stress and hormone induction. We showed that overexpression of FmSPL2 in transgenic Nicotiana tabacum L. resulted in taller plants, shorter root length, increased root number, rounded leaves, and earlier flowering time.ConclusionsWe identified 36 SPL genes, which were classified into seven subfamilies based on sequence analysis. FmSPL2 was selected for subsequent heterologous expression by analysis of expression patterns in various tissues and under abiotic stress and hormone induction, and significant phenotypic changes were observed in the transgenic Nicotiana tabacum L. These results provide insight into the evolutionary origin and biological significance of plant SPL. The aim of this study was to lay the foundation for the genetic improvement of Fraxinus mandshurica and the subsequent functional analysis of FmSPL2.

Overexpression of PSY1 increases fruit skin and flesh carotenoid content and reveals associated transcription factors in apple (Malus × domestica).

Knowledge of the transcriptional regulation of the carotenoid metabolic pathway is still emerging and here, we have misexpressed a key biosynthetic gene in apple to highlight potential transcriptional regulators of this pathway. We overexpressed phytoene synthase (PSY1), which controls the key rate-limiting biosynthetic step, in apple and analyzed its effects in transgenic fruit skin and flesh using two approaches. Firstly, the effects of PSY overexpression on carotenoid accumulation and gene expression was assessed in fruit at different development stages. Secondly, the effect of light exclusion on PSY1-induced fruit carotenoid accumulation was examined. PSY1 overexpression increased carotenoid content in transgenic fruit skin and flesh, with beta-carotene being the most prevalent carotenoid compound. Light exclusion by fruit bagging reduced carotenoid content overall, but carotenoid content was still higher in bagged PSY fruit than in bagged controls. In tissues overexpressing PSY1, plastids showed accelerated chloroplast to chromoplast transition as well as high fluorescence intensity, consistent with increased number of chromoplasts and carotenoid accumulation. Surprisingly, the expression of other carotenoid pathway genes was elevated in PSY fruit, suggesting a feed-forward regulation of carotenogenesis when this enzyme step is mis-expressed. Transcriptome profiling of fruit flesh identified differentially expressed transcription factors (TFs) that also were co-expressed with carotenoid pathway genes. A comparison of differentially expressed genes from both the developmental series and light exclusion treatment revealed six candidate TFs exhibiting strong correlation with carotenoid accumulation. This combination of physiological, transcriptomic and metabolite data sheds new light on plant carotenogenesis and TFs that may play a role in regulating apple carotenoid biosynthesis.

Comparative transcriptomics analysis of developing peanut (Arachis hypogaea L.) pods reveals candidate genes affecting peanut seed size.

Pod size is one of the most important agronomic features of peanuts, which directly affects peanut yield. Studies on the regulation mechanism underpinning pod size in cultivated peanuts remain hitherto limited compared to model plant systems. To better understand the molecular elements that underpin peanut pod development, we conducted a comprehensive analysis of chronological transcriptomics during pod development in four peanut accessions with similar genetic backgrounds, but varying pod sizes. Several plant transcription factors, phytohormones, and the mitogen-activated protein kinase (MAPK) signaling pathways were significantly enriched among differentially expressed genes (DEGs) at five consecutive developmental stages, revealing an eclectic range of candidate genes, including PNC, YUC, and IAA that regulate auxin synthesis and metabolism, CYCD and CYCU that regulate cell differentiation and proliferation, and GASA that regulates seed size and pod elongation via gibberellin pathway. It is plausible that MPK3 promotes integument cell division and regulates mitotic activity through phosphorylation, and the interactions between these genes form a network of molecular pathways that affect peanut pod size. Furthermore, two variant sites, GCP4 and RPPL1, were identified which are stable at the QTL interval for seed size attributes and function in plant cell tissue microtubule nucleation. These findings may facilitate the identification of candidate genes that regulate pod size and impart yield improvement in cultivated peanuts.

Multilevel regulation of anthocyanin-promoting R2R3-MYB transcription factors in plants.

Anthocyanins are common secondary metabolites in plants that confer red, blue, and purple colorations in plants and are highly desired by consumers for their visual appearance and nutritional quality. In the last two decades, the anthocyanin biosynthetic pathway and transcriptional regulation of anthocyanin biosynthetic genes (ABGs) have been well characterized in many plants. From numerous studies on model plants and horticultural crops, many signaling regulators have been found to control anthocyanin accumulation via regulation of anthocyanin-promoting R2R3-MYB transcription factors (so-called R2R3-MYB activators). The regulatory mechanism of R2R3-MYB activators is mediated by multiple environmental factors (e.g., light, temperature) and internal signals (e.g., sugar, ethylene, and JA) in complicated interactions at multiple levels. Here, we summarize the transcriptional control of R2R3-MYB activators as a result of natural variations in the promoter of their encoding genes, upstream transcription factors and epigenetics, and posttranslational modifications of R2R3-MYB that determine color variations of horticultural plants. In addition, we focus on progress in elucidating the integrated regulatory network of anthocyanin biosynthesis mediated by R2R3-MYB activators in response to multiple signals. We also highlight a few gene cascade modules involved in the regulation of anthocyanin-related R2R3-MYB to provide insights into anthocyanin production in horticultural plants.

A transcriptomic-guided strategy used in identification of a wheat rust pathogen target and modification of the target enhanced host resistance to rust pathogens.

Transcriptional reprogramming is an essential feature of plant immunity and is governed by transcription factors (TFs) and co-regulatory proteins associated with discrete transcriptional complexes. On the other hand, effector proteins from pathogens have been shown to hijack these vast repertoires of plant TFs. Our current knowledge of host genes' role (including TFs) involved in pathogen colonization is based on research employing model plants such as Arabidopsis and rice with minimal efforts in wheat rust interactions. In this study, we begun the research by identifying wheat genes that benefit rust pathogens during infection and editing those genes to provide wheat with passive resistance to rust. We identified the wheat MYC4 transcription factor (TF) located on chromosome 1B (TaMYC4-1B) as a rust pathogen target. The gene was upregulated only in susceptible lines in the presence of the pathogens. Down-regulation of TaMYC4-1B using barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) in the susceptible cultivar Chinese Spring enhanced its resistance to the stem rust pathogen. Knockout of the TaMYC4-1BL in Cadenza rendered new resistance to races of stem, leaf, and stripe rust pathogens. We developed new germplasm in wheat via modifications of the wheat TaMYC4−1BL transcription factor.

Tartary buckwheat FtMYB30 transcription factor improves the salt/drought tolerance of transgenic Arabidopsis in an ABA-dependent manner.

Drought and high salinity affect plant growth, development, yield, and quality. MYB transcription factors (TFs) in plants play an indispensable regulatory role in resisting adverse stress. In this study, screening and functional validation of the TF FtMYB30, which can respond extensively to abiotic stress and abscisic acid (ABA), was achieved in Tartary buckwheat. FtMYB30, one of the SG22 (sub-group 22) family of R2R3-MYB TFs, localized in the nucleus and had transcriptional activation activity. Under drought and salt stress, FtMYB30 overexpression reduced the oxidative damage in transgenic plants by increasing the activity of proline content and antioxidant enzymes and significantly upregulate the expression of RD29A, RD29B, and Cu/ZnSOD, thereby enhancing drought/salt tolerance in transgenic Arabidopsis. Additionally, overexpression of FtMYB30 can reduce the sensitivity of transgenic plants to ABA. Moreover, AtRCAR1/2/3 and AtMPK6 directly interact with the FtMYB30 TF, possibly through the crosstalk between MAPKs (mitogen-activated protein kinases) and the ABA signaling pathway. Taken together, these results suggest that FtMYB30, as a positive regulator, mediates plant tolerance to salt and drought through an ABA-dependent signaling pathway.

Characterization of Virus-Inducible Orchid Argonaute 5b Promoter and Its Functional Characterization in Nicotiana benthamiana during Virus Infection.

Plant ARGONAUTES (AGOs) play a significant role in the defense against viral infection. Previously, we have demonstrated that AGO5s encoded in Phalaenopsis aphrodite subsp. formosana (PaAGO5s) took an indispensable part in defense against major viruses. To understand the underlying defense mechanism, we cloned PaAGO5s promoters (pPaAGO5s) and analyzed their activity in transgenic Nicotiana benthamiana using β-glucuronidase (GUS) as a reporter gene. GUS activity analyses revealed that during Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) infections, pPaAGO5b activity was significantly increased compared to pPaAGO5a and pPaAGO5c. Analysis of pPaAGO5b 5′-deletion revealed that pPaAGO5b_941 has higher activity during virus infection. Further, yeast one-hybrid analysis showed that the transcription factor NbMYB30 physically interacted with pPaAGO5b_941 to enhance its activity. Overexpression and silencing of NbMYB30 resulted in up- and downregulation of GUS expression, respectively. Exogenous application and endogenous measurement of phytohormones have shown that methyl jasmonate and salicylic acid respond to viral infections. NbMYB30 overexpression and its closest related protein, PaMYB30, in P. aphrodite subsp. formosana reduced CymMV accumulation in P. aphrodite subsp. formosana. Based on these discoveries, this study uncovers the interaction between virus-responsive promoter and the corresponding transcription factor in plants.

A Transcription Factor SlNAC10 Gene of Suaeda liaotungensis Regulates Proline Synthesis and Enhances Salt and Drought Tolerance.

The NAC (NAM, ATAF1/2, and CUC2) transcription factors are one of the largest families of transcription factors in plants and play an important role in plant development and the response to adversity. In this study, we cloned a new NAC gene, SlNAC10, from the halophyte Suaeda liaotungensis K. The gene has a total length of 1584 bp including a complete ORF of 1107 bp that encodes 369 amino acids. The SlNAC10-GFP fusion protein is located in the nucleus and SlNAC10 has a transcription activation structural domain at the C-terminus. We studied the expression characteristics of SlNAC10 and found that it was highest in the leaves of S. liaotungensis and induced by drought, salt, cold, and abscisic acid (ABA). To analyze the function of SlNAC10 in plants, we obtained SlNAC10 transgenic Arabidopsis. The growth characteristics and physiological indicators of transgenic Arabidopsis were measured under salt and drought stress. The transgenic Arabidopsis showed obvious advantages in the root length and survival rate; chlorophyll fluorescence levels; and the antioxidant enzyme superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities, and the proline content was higher than that of the wild-type (WT) Arabidopsis, whereas the relative electrolyte leakage and malondialdehyde (MDA) content were lower than those of the wild-type Arabidopsis. We explored the regulatory role of SlNAC10 on proline synthesis-related enzyme genes and found that SlNAC10 binds to the AtP5CS1, AtP5CS2, and AtP5CR promoters and regulates their downstream gene transcription. To sum up, SlNAC10 as a transcription factor improves salt and drought tolerance in plants possibly by regulating proline synthesis.

Genome-wide identification and comprehensive analyses of NAC transcription factor gene family and expression analysis under Fusarium kyushuense and drought stress conditions in Passiflora edulis.

The NAC gene family is one of the largest plant transcription factors (TFs) families and plays important roles in plant growth, development, metabolism, and biotic and abiotic stresses. However, NAC gene family has not been reported in passion fruit (Passiflora edulis). In this study, a total of 105 NAC genes were identified in the passion fruit genome and were unevenly distributed across all nine-passion fruit chromomere, with a maximum of 48 PeNAC genes on chromosome one. The physicochemical features of all 105 PeNAC genes varied including 120 to 3,052 amino acids, 3 to 8 conserved motifs, and 1 to 3 introns. The PeNAC genes were named (PeNAC001–PeNAC105) according to their chromosomal locations and phylogenetically grouped into 15 clades (NAC-a to NAC-o). Most PeNAC proteins were predicted to be localized in the nucleus. The cis-element analysis indicated the possible roles of PeNAC genes in plant growth, development, light, hormones, and stress responsiveness. Moreover, the PeNAC gene duplications including tandem (11 gene pairs) and segmental (12 gene pairs) were identified and subjected to purifying selection. All PeNAC proteins exhibited similar 3D structures, and a protein–protein interaction network analysis with known Arabidopsis proteins was predicted. Furthermore, 17 putative ped-miRNAs were identified to target 25 PeNAC genes. Potential TFs including ERF, BBR-BPC, Dof, and bZIP were identified in promoter region of all 105 PeNAC genes and visualized in a TF regulatory network. GO and KEGG annotation analysis exposed that PeNAC genes were related to different biological, molecular, and cellular terms. The qRT-PCR expression analysis discovered that most of the PeNAC genes including PeNAC001, PeNAC003, PeNAC008, PeNAC028, PeNAC033, PeNAC058, PeNAC063, and PeNAC077 were significantly upregulated under Fusarium kyushuense and drought stress conditions compared to controls. In conclusion, these findings lay the foundation for further functional studies of PeNAC genes to facilitate the genetic improvement of plants to stress resistance.

Plant-DTI: Extending the landscape of TF protein and DNA interaction in plants by a machine learning-based approach.

As a sessile organism, plants hold elaborate transcriptional regulatory systems that allow them to adapt to variable surrounding environments. Current understanding of plant regulatory mechanisms is greatly constrained by limited knowledge of transcription factor (TF)–DNA interactions. To mitigate this problem, a Plant-DTI predictor (Plant DBD-TFBS Interaction) was developed here as the first machine-learning model that covered the largest experimental datasets of 30 plant TF families, including 7 plant-specific DNA binding domain (DBD) types, and their transcription factor binding sites (TFBSs). Plant-DTI introduced a novel TFBS feature construction, called TFBS base-preference, which enhanced the specificity of TFBS to DBD types. The proposed model showed better predictive performance with the TFBS base-preference than the simple binary representation. Plant-DTI was validated with 22 independent ChIP-seq datasets. It accurately predicted the measured DBD-TFBS pairs along with their TFBS motifs, and effectively predicted interactions of other TFs containing similar DBD types. Comparing to the existing state-of-art methods, Plant-DTI prediction showed a figure of merit in sensitivity and specificity with respect to the position weight matrix (PWM) and TSPTFBS methods. Finally, the proposed Plant-DTI model helped to fill the knowledge gap in the regulatory mechanisms of the cassava sucrose synthase 1 gene (MeSUS1). Plant-DTI predicted MeERF72 as a regulator of MeSUS1 in consistence with the yeast one-hybrid (Y1H) experiment. Taken together, Plant-DTI would help facilitate the prediction of TF-TFBS and TF-target gene (TG) interactions, thereby accelerating the study of transcriptional regulatory systems in plant species.

Isolation and Characterization of an LBD Transcription Factor CsLBD39 from Tea Plant (Camellia sinensis) and Its Roles in Modulating Nitrate Content by Regulating Nitrate-Metabolism-Related Genes.

Nitrate nitrogen is an important nitrogen source for tea plants’ growth and development. LBD transcription factors play important roles in response to the presence of nitrate in plants. The functional study of LBD transcription factors in tea plants remains limited. In this study, the LBD family gene CsLBD39 was isolated and characterized from tea plants. Sequence analysis indicated that CsLBD39 contained a highly conserved CX2CX6CX3CX domain. The phylogenetic tree assay showed that CsLBD39 belonged to class II subfamily of the LBD family. CsLBD39 was highly expressed in flowers and root; we determined that its expression could be induced by nitrate treatment. The CsLBD39 protein was located in the nucleus and has transcriptional activation activity in yeast. Compared with the wild type, overexpression of CsLBD39 gene in Arabidopsis resulted in smaller rosettes, shorter main roots, reduced lateral roots and lower plant weights. The nitrate content and the expression levels of genes related to nitrate transport and regulation were decreased in transgenic Arabidopsis hosting CsLBD39 gene. Compared with the wild type, CsLBD39 overexpression in transgenic Arabidopsis had smaller cell structure of leaves, shorter diameter of stem cross section, and slender and compact cell of stem longitudinal section. Under KNO3 treatment, the contents of nitrate, anthocyanins, and chlorophyll in leaves, and the content of nitrate in roots of Arabidopsis overexpressing CsLBD39 were reduced, the expression levels of nitrate transport and regulation related genes were decreased. The results revealed that CsLBD39 may be involved in nitrate signal transduction in tea plants as a negative regulator and laid the groundwork for future studies into the mechanism of nitrate response.

Genome-Wide Identification and Characterization of YABBY Gene Family in Juglans regia and Juglans mandshurica

The YABBY gene family is a plant transcription factor that exists in all seed plants. YABBY family members have been studied extensively in various plants and were to play significant roles in plant growth and development. Juglans, especially walnuts, are important economic tree species that are widely distributed worldwide. However, the identification and related research of YABBY in Juglans have not been reported to date. In this study, we identified 19 YABBY genes from two Juglans species, namely, J. regia and J. mandshurica. Ten JrYABBY genes and nine JmYABBY genes were divided into five subfamilies (YAB1/3, YAB2, INO, CRC, and YAB5). Sequence analysis revealed that all encoded YABBY protein sequences had a highly conserved YABBY and C2C2 zinc-finger domains. An analysis of the assumed cis-acting elements revealed that JrYABBY and JmYABBY genes were deeply involved in phytohormone and light responses. Further, gene expression pattern analysis suggested that most walnut YABBY genes were likely involved in peel and flower development and responses to biotic stress. This study not only suppled novel insights into the evolutionary basis of YABBY gene families in Juglans, but also provided clues for the further functional verification and investigation of YABBY genes in other tree species.

Genome-wide investigation of AP2/ERF gene family in the desert legume Eremosparton songoricum: Identification, classification, evolution, and expression profiling under drought stress.

Eremosparton songoricum (Litv.) Vass. is a rare leafless legume shrub endemic to central Asia which grows on bare sand. It shows extreme drought tolerance and is being developed as a model organism for investigating morphological, physiological, and molecular adaptations to harsh desert environments. APETALA2/Ethylene Responsive Factor (AP2/ERF) is a large plant transcription factor family that plays important roles in plant responses to various biotic and abiotic stresses and has been extensively studied in several plants. However, our knowledge on the AP2/ERF family in legume species is limited, and no respective study was conducted so far on the desert shrubby legume E. songoricum. Here, 153 AP2/ERF genes were identified based on the E. songoricum genome data. EsAP2/ERFs covered AP2 (24 genes), DREB (59 genes), ERF (68 genes), and Soloist (2 genes) subfamilies, and lacked canonical RAV subfamily genes based on the widely used classification method. The DREB and ERF subfamilies were further divided into A1–A6 and B1–B6 groups, respectively. Protein motifs and exon-intron structures of EsAP2/ERFs were also examined, which matched the subfamily/group classification. Cis-acting element analysis suggested that EsAP2/ERF genes shared many stress- and hormone-related cis-regulatory elements. Moreover, the gene numbers and the ratio of each subfamily and the intron-exon structures were systematically compared with other model plants ranging from algae to angiosperms, including ten legumes. Our results supported the view that AP2 and ERF evolved early and already existed in algae, whereas RAV and DREB began to appear in moss species. Almost all plant AP2 and Soloist genes contained introns, whereas most DREB and ERF genes did not. The majority of EsAP2/ERFs were induced by drought stress based on RNA-seq data, EsDREBs were highly induced and had the largest number of differentially expressed genes in response to drought. Eight out of twelve representative EsAP2/ERFs were significantly up-regulated as assessed by RT-qPCR. This study provides detailed insights into the classification, gene structure, motifs, chromosome distribution, and gene expression of AP2/ERF genes in E. songoricum and lays a foundation for better understanding of drought stress tolerance mechanisms in legume plants. Moreover, candidate genes for drought-resistant plant breeding are proposed.

Motif models proposing independent and interdependent impacts of nucleotides are related to high and low affinity transcription factor binding sites in Arabidopsis

Position weight matrix (PWM) is the traditional motif model representing the transcription factor (TF) binding sites. It proposes that the positions contribute independently to TFs binding affinity, although this hypothesis does not fit the data perfectly. This explains why PWM hits are missing in a substantial fraction of ChIP-seq peaks. To study various modes of the direct binding of plant TFs, we compiled the benchmark collection of 111 ChIP-seq datasets for Arabidopsis thaliana, and applied the traditional PWM, and two alternative motif models BaMM and SiteGA, proposing the dependencies of the positions. The variation in the stringency of the recognition thresholds for the models proposed that the hits of PWM, BaMM, and SiteGA models are associated with the sites of high/medium, any, and low affinity, respectively. At the medium recognition threshold, about 60% of ChIP-seq peaks contain PWM hits consisting of conserved core consensuses, while BaMM and SiteGA provide hits for an additional 15% of peaks in which a weaker core consensus is compensated through intra-motif dependencies. The presence/absence of these dependencies in the motifs of alternative/traditional models was confirmed by the dependency logo DepLogo visualizing the position-wise partitioning of the alignments of predicted sites. We exemplify the detailed analysis of ChIP-seq profiles for plant TFs CCA1, MYC2, and SEP3. Gene ontology (GO) enrichment analysis revealed that among the three motif models, the SiteGA had the highest portions of genes with the significantly enriched GO terms among all predicted genes. We showed that both alternative motif models provide for traditional PWM greater extensions in predicted sites for TFs MYC2/SEP3 with condition/tissue specific functions, compared to those for TF CCA1 with housekeeping functions. Overall, the combined application of standard and alternative motif models is beneficial to detect various modes of the direct TF-DNA interactions in the maximal portion of ChIP-seq loci.

GmWRKY21, a Soybean WRKY Transcription Factor Gene, Enhances the Tolerance to Aluminum Stress in Arabidopsis thaliana.

The WRKY transcription factors (TFs) are one of the largest families of TFs in plants and play multiple roles in plant growth and development and stress response. In this study, GmWRKY21 encoding a WRKY transcription factor was functionally characterized in Arabidopsis and soybean. The GmWRKY21 protein containing a highly conserved WRKY domain and a C2H2 zinc-finger structure is located in the nucleus and has the characteristics of transcriptional activation ability. The GmWRKY21 gene presented a constitutive expression pattern rich in the roots, leaves, and flowers of soybean with over 6-fold of relative expression levels and could be substantially induced by aluminum stress. As compared to the control, overexpression of GmWRKY21 in Arabidopsis increased the root growth of seedlings in transgenic lines under the AlCl3 concentrations of 25, 50, and 100 μM with higher proline and lower MDA accumulation. The results of quantitative real-time polymerase chain reaction (qRT-PCR) showed that the marker genes relative to aluminum stress including ALMT, ALS3, MATE, and STOP1 were induced in GmWRKY21 transgenic plants under AlCl3 treatment. The stress-related genes, such as KIN1, COR15A, COR15B, COR47, GLOS3, and RD29A, were also upregulated in GmWRKY21 transgenic Arabidopsis under aluminum stress. Similarly, stress-related genes, such as GmCOR47, GmDREB2A, GmMYB84, GmKIN1, GmGST1, and GmLEA, were upregulated in hair roots of GmWRKY21 transgenic plants. In summary, these results suggested that the GmWRKY21 transcription factor may promote the tolerance to aluminum stress mediated by the pathways regulating the expression of the acidic aluminum stress-responsive genes and abiotic stress-responsive genes.

GsMYB7 encoding a R2R3-type MYB transcription factor enhances the tolerance to aluminum stress in soybean (Glycine max L.)

BackgroundMYB transcription factor (TF) is one of the largest families of TFs in plants and play essential roles in plant growth and development, and is involved in responses to biological and abiotic stress. However, there are few reports on GsMYB7 gene in soybean under aluminum acid stress, and its regulatory mechanism remains unclear.ResultsThe GsMYB7 protein is localized in the nucleus and has transcriptional activation ability. Quantitative real-time PCR (qRT-PCR) results showed that GsMYB7 held a constitutive expression pattern rich in roots. When AlCl3 concentration was 25 µM, the total root surface area (SA) of GsMYB7 transgenic lines were 34.97% higher than that of wild-type Huachun 6 (HC6). While the accumulation of Al3+ in root tip of transgenic plants after aluminum treatment was 17.39% lower than that of wild-type. RNA-sequencing analysis indicated that over 1181 genes were regulated by GsMYB7 and aluminum stress. Among all the regulated genes, the expression levels of glutathione peroxidase, protein kinase, cytochrome and other genes in the transgenic lines were significantly higher than those in wild type by acidic aluminum stress. The bioinformatics and qRT-PCR results showed that 9 candidate genes were induced under the treatments of acidic aluminum stress which were indirectly and/or directly regulated by GsMYB7. After AlCl3 treatments, the transcripts of these genes in GsMYB7 transgenic seedlings were significantly higher than those of wide-type HC6.ConclusionsThe results suggested that GsMYB7 may enhance soybean tolerance to acidic aluminum stress by regulating the downstream genes.