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The WRKY gene family is ubiquitously distributed in plants, serving crucial functions in stress responses. Nevertheless, the structural organization and evolutionary dynamics of WRKY genes in cotton have not been fully elucidated. In this study, a total of 112, 119, 217, and 222 WRKY genes were identified in Gossypium arboreum, Gossypium raimondii, Gossypium hirsutum, and Gossypium barbadense, respectively. These 670 WRKY genes were categorized into seven distinct subgroups and unequally distributed across chromosomes. Examination of conserved motifs, domains, cis-acting elements, and gene architecture collectively highlighted the evolutionary conservation and divergence within the WRKY gene family in cotton. Analysis of synteny and collinearity further confirmed instances of expansion, duplication, and loss events among WRKY genes during cotton evolution. Furthermore, GhWRKY31 transgenic Arabidopsis exhibited heightened germination rates and longer root lengths under drought and salt stress. Silencing GhWRKY31 in cotton led to reduced levels of ABA, proline, POD, and SOD, along with downregulated expression of stress-responsive genes. Yeast one-hybrid and molecular docking assays confirmed the binding capacity of GhWRKY31 to the W box of GhABF1, GhDREB2, and GhRD29. The findings collectively offer a systematic and comprehensive insight into the evolutionary patterns of cotton WRKYs, proposing a suitable regulatory framework for developing cotton cultivars with enhanced resilience to drought and salinity stress. [ABSTRACT FROM AUTHOR]

Aureochromes (AUREOs) are unique blue light receptors and transcription factors found only in stramenopile algae. While each of the four AUREOs identified in the diatom Phaeodactylum tricornutum may have a specific function, PtAUREO1a has been shown to have a strong impact on overall gene regulation, when light changes from red to blue light conditions. Despite its significance, the molecular mechanism of PtAUREO1a is largely unexplored. To comprehend the overall process of gene regulation by PtAUREO1a, we conducted a series of in vitro and in vivo experiments, including pull-down assays, yeast one-hybrid experiments, and phenotypical characterization using recombinant PtAUREOs and diatom mutant lines expressing a modified PtAureo1a gene. We describe the distinct light absorption properties of four PtAUREOs and the formation of all combinations of their potential dimers. We demonstrate the capability of PtAUREO1a and 1b to activate the genes, diatom-specific cyclin 2 , PtAureo1a , and PtAureo1c under both light and dark conditions. Using mutant lines expressing a modified PtAUREO1a protein with a considerably reduced light absorption, we found novel evidence that PtAUREO1a regulates the expression of PtLHCF15, which is essential for red light acclimation. Based on current knowledge, we present a working model of PtAUREO1a gene regulation properties. [ABSTRACT FROM AUTHOR]

Aliphatic glucosinolates are a large group of plant secondary metabolites characteristic of Brassicaceae , including the model plant Arabidopsis. The diverse and complex degradation products of aliphatic glucosinolates contribute to plant responses to herbivory, pathogen attack, and environmental stresses. Most of the biosynthesis genes in the aliphatic glucosinolate pathway have been cloned in Arabidopsis, and the research focus has recently shifted to the regulatory mechanisms controlling aliphatic glucosinolate accumulation. Up till now, more than 40 transcriptional regulators have been identified as regulating the aliphatic glucosinolate pathway, but many more novel regulators likely remain to be discovered based on research evidence over the past decade. In the current study, we took a systemic approach to functionally test 155 candidate transcription factors in Arabidopsis identified by yeast one-hybrid assay, and successfully validated at least 30 novel regulators that could significantly influence the accumulation of aliphatic glucosinolates in our experimental set-up. We also showed that the regulators of the aliphatic glucosinolate pathway have balanced positive and negative effects, and glucosinolate metabolism and plant development can be coordinated. Our work is the largest scale effort so far to validate transcriptional regulators of a plant secondary metabolism pathway, and provides new insights into how the highly diverse plant secondary metabolism is regulated at the transcriptional level. [ABSTRACT FROM AUTHOR]

The WRKY gene family is ubiquitously distributed in plants, serving crucial functions in stress responses. Nevertheless, the structural organization and evolutionary dynamics of WRKY genes in cotton have not been fully elucidated. In this study, a total of 112, 119, 217, and 222 WRKY genes were identified in Gossypium arboreum, Gossypium raimondii, Gossypium hirsutum, and Gossypium barbadense, respectively. These 670 WRKY genes were categorized into seven distinct subgroups and unequally distributed across chromosomes. Examination of conserved motifs, domains, cis-acting elements, and gene architecture collectively highlighted the evolutionary conservation and divergence within the WRKY gene family in cotton. Analysis of synteny and collinearity further confirmed instances of expansion, duplication, and loss events among WRKY genes during cotton evolution. Furthermore, GhWRKY31 transgenic Arabidopsis exhibited heightened germination rates and longer root lengths under drought and salt stress. Silencing GhWRKY31 in cotton led to reduced levels of ABA, proline, POD, and SOD, along with downregulated expression of stress-responsive genes. Yeast one-hybrid and molecular docking assays confirmed the binding capacity of GhWRKY31 to the W box of GhABF1, GhDREB2, and GhRD29. The findings collectively offer a systematic and comprehensive insight into the evolutionary patterns of cotton WRKYs, proposing a suitable regulatory framework for developing cotton cultivars with enhanced resilience to drought and salinity stress.

A comparative metabolomic analysis was performed using purple potato tubers and derived red mutant tubers to investigate the regulation of anthocyanin biosynthesis. The metabolome analysis revealed 19 key differential metabolites including 10 flavonoids and 4 nucleotides. As a differential metabolite, pelargonidin was responsible for the mutant, and other metabolites had a correlation with it. To further analyse the regulatory mechanism of the differential metabolites, correlation analyses of metabolome and transcriptome were done. Among the differential metabolites, seven flavonoids and F3′5′H were in the flavonoid metabolic pathway. F3′5′H is responsible for the anthocyanin biosynthesis branch in potato tubers. To further analyse the regulatory mechanism of F3′5′H, the transcriptome analysis identified 63 differentially expressed genes, which highly correlated with F3'5'H. Furthermore, correlations among the key differential metabolites and the differentially expressed genes indicated that 11 differential genes and 10 differential metabolites presented 76 significant correlations. These 11 genes mainly encoded serine/threonine protein kinase, UMP-CMP kinase, MADS-box transcription factor, and WD repeat-containing protein. The 10 differential metabolites were involved in pelargonidin, luteolin, kaempferol-3-O-rutinoside, eriodictyol-7-O-glucoside, and uridine monophosphate. Through yeast one-hybrid experiments, the proteins interacting with the promoter of F3'5'H were screened: ethylene-responsive transcription factor ERF011, gibberellin-regulated protein 9-like, and serine-threonine protein kinase, transcription factor LHW bHLH. These results will provide valuable insights into the regulation mechanism of the anthocyanin biosynthesis branch in potatoes. [ABSTRACT FROM AUTHOR]

The indolizidine alkaloid-swainsonine (SW) is the main toxic component of locoweeds and the main cause of locoweed poisoning in grazing animals. The endophytic fungi, Alternaria Section Undifilum spp., are responsible for the biosynthesis of SW in locoweeds. The swnK gene is a multifunctional complex enzyme encoding gene in fungal SW biosynthesis, and its encoding product plays a key role in the multistep catalytic synthesis of SW by fungi using pipecolic acid as a precursor. However, the transcriptional regulation mechanism of the swnK gene is still unclear. To identify the transcriptional regulators involved in the swnK gene in endophytic fungi of locoweeds, we first analyzed the upstream non-coding region of the swnK gene in the A. oxytropis UA003 strain and predicted its high transcriptional activity region combined with dual-luciferase reporter assay. Then, a yeast one-hybrid library of A. oxytropis UA003 strain was constructed, and the transcriptional regulatory factors that may bind to the high-transcriptional activity region of the upstream non-coding region of the swnK gene were screened by this system. The results showed that the high transcriptional activity region was located at −656 bp and −392 bp of the upstream regulatory region of the swnK gene. A total of nine candidate transcriptional regulator molecules, including a C2H2 type transcription factor, seven annotated proteins, and an unannotated protein, were screened out through the Y1H system, which were bound to the upstream high transcriptional activity region of the swnK gene. This study provides new insight into the transcriptional regulation of the swnK gene and lays the foundation for further exploration of the regulatory mechanisms of SW biosynthesis in fungal endophytic locoweeds. [ABSTRACT FROM AUTHOR]

BRANCHED1 (BRC1) is a crucial member of the TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) gene family and is well known for playing a central role in shoot branching by controlling buds' paradormancy. However, the expression characteristics and molecular regulatory mechanism of BRC1 during blueberry bud dormancy are unclear. To shed light on these topics, shoots of three blueberry cultivars with different chilling requirements (CRs) were decapitated in summer to induce paradormancy release and subjected to different levels of chilling in winter to induce endodormancy release. The results showed that the high-CR cultivar 'Chandler' had the strongest apical dominance among the three cultivars; additionally, the expression of VcTCP18, which is homologous to BRC1, was the highest under both the decapitation treatment and low-temperature treatment. The 'Emerald' cultivar, with a low CR, demonstrated the opposite trend. These findings suggest that VcTCP18 plays a negative regulatory role in bud break and that there may be a correlation between the CR and tree shape. Through yeast 1-hybrid (Y1H) assays, we finally screened 21 upstream regulatory genes, including eight transcription factors: zinc-finger homeodomain protein 1/4/5/9, MYB4, AP2-like ethylene-responsive transcription factor AINTEGUMENTA (ANT), ASIL2-like, and bHLH035. It was found that these upstream regulatory genes positively or negatively regulated the expression of VcTCP18 based on the transcriptome expression profile. In summary, this study enriched our understanding of the regulatory network of BRCl during bud dormancy and provided new insights into the function of BRC1. [ABSTRACT FROM AUTHOR]

[Objective] The NLPS family of transcription factors were identified as nitrate-responsive ciselement (NRE)-binding proteins, which functioned as transcriptional activators in the nitrate-regulated expression of downstream genes. Transporters of ZmSTP1 and ZmAAP2 played important roles in the transport and unloading of C-N assimilates in maize. In addition, both ZmSTPI and ZmAAP2 contained NRE cis-elements in the promoter regions. The present study aimed to determine whether NLP gene family in maize were NRE binding proteins of ZmSTPI and ZmAAP2 promoters. [Method] Maize B73 as experimental material was used to explore the interaction between ZmNLP3, ZmN- LP4, ZmSTPI and ZmAAP2 by yeast one-hybrid system. [Result] The analysis of 2000 bp sequences upstream of the transcription start sites of ZmSTPI and ZmAAP2 genes by Plantcare showed that NRE cis-elements were located in -577 to -589 bp and 909 to 921 bp re- gions upstream of the transcription start sites of the two genes. The structures were 5'-ATTTAATACTCA-3' and 5'-ATATATAACTCA-3'. AbA' gene expression detection of bait strains showed that the lowest AbA concentration that could inhibit the background expression of bait strains YIH(pAbAi-ZmSTPI) and Y1H (pAbAi-ZmAAP2) was 200 ng/mL. The pGADT7-ZmNLP3/4 were transformed into the Y1H (pAbAi-ZmSTPI) and YIH(pAbAi-ZmAAP2) strains, and all strains could grow normally on control medium (SD/-Leu) and screening medium with AbA concentration of 200 ng/mL.. [Conclusion] ZmNLP3 and ZmNLP4 can interact with ZmSTP1 and ZmAAP2. ZmNLP3 and ZmNLP4 are binding proteins of NRE elements in the promoter region of ZmSTPI and ZmAAP2. The results of this study not only contribute to the further understanding of nitrate signal transduction involving members of ZmNLPs family, but also provide the theory basis for molecu- lar breeding for increasing maize yield and nitrogen efficiency. [ABSTRACT FROM AUTHOR]

The transcription factors of basic leucine zipper (bZIP) family genes play significant roles in stress response as well as growth and development in plants. However, little is known about the bZIP gene family in Chinese chestnut (Castanea mollissima Blume). To better understand the characteristics of bZIPs in chestnut and their function in starch accumulation, a series of analyses were performed including phylogenetic, synteny, co-expression and yeast one-hybrid analyses. Totally, we identified 59 bZIP genes that were unevenly distributed in the chestnut genome and named them CmbZIP01 to CmbZIP59. These CmbZIPs were clustered into 13 clades with clade-specific motifs and structures. A synteny analysis revealed that segmental duplication was the major driving force of expansion of the CmbZIP gene family. A total of 41 CmbZIP genes had syntenic relationships with four other species. The results from the co-expression analyses indicated that seven CmbZIPs in three keymodules may be important in regulating starch accumulation in chestnut seeds. Yeast one-hybrid assays showed that transcription factors CmbZIP13 and CmbZIP35 might participate in starch accumulation in the chestnut seed by binding to the promoters of CmISA2 and CmSBE1_2, respectively. Our study provided basic information on CmbZIP genes, which can be utilized in future functional analysis and breeding studies. [ABSTRACT FROM AUTHOR]

Introduction: Phosphorus (P) is one of the most important nutrient elements for plant growth and development. Under P starvation, arbuscular mycorrhizal (AM) fungi can promote phosphate (Pi) uptake and homeostasis within host plants. However, the underlying mechanisms by which AM fungal symbiont regulates the AM symbiotic Pi acquisition from soil under P starvation are largely unknown. Here, we identify a HLH domain containing transcription factor RiPho4 from Rhizophagus irregularis. Methods: To investigate the biological functions of the RiPho4, we combined the subcellular localization and Yeast One-Hybrid (Y1H) experiments in yeasts with gene expression and virus-induced gene silencing approach during AM symbiosis. Results: The approach during AM symbiosis. The results indicated that RiPho4 encodes a conserved transcription factor among different fungi and is induced during the in planta phase. The transcription of RiPho4 is significantly upregulated by P starvation. The subcellular localization analysis revealed that RiPho4 is located in the nuclei of yeast cells during P starvation. Moreover, knock-down of RiPho4 inhibits the arbuscule development and mycorrhizal Pi uptake under low Pi conditions. Importantly, RiPho4 can positively regulate the downstream components of the phosphate (PHO) pathway in R. irregularis. Discussion: In summary, these new findings reveal that RiPho4 acts as a transcriptional activator in AM fungus to maintain arbuscule development and regulate Pi uptake and homeostasis in the AM symbiosis during Pi starvation. [ABSTRACT FROM AUTHOR]

Copyright of Acta Agriculturae Zhejiangensis is the property of Acta Agriculturae Zhejiangensis Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Acta Agriculturae Zhejiangensis is the property of Acta Agriculturae Zhejiangensis Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The indolizidine alkaloid-swainsonine (SW) is the main toxic component of locoweeds and the main cause of locoweed poisoning in grazing animals. The endophytic fungi, Alternaria Section Undifilum spp., are responsible for the biosynthesis of SW in locoweeds. The swnK gene is a multifunctional complex enzyme encoding gene in fungal SW biosynthesis, and its encoding product plays a key role in the multistep catalytic synthesis of SW by fungi using pipecolic acid as a precursor. However, the transcriptional regulation mechanism of the swnK gene is still unclear. To identify the transcriptional regulators involved in the swnK gene in endophytic fungi of locoweeds, we first analyzed the upstream non-coding region of the swnK gene in the A. oxytropis UA003 strain and predicted its high transcriptional activity region combined with dual-luciferase reporter assay. Then, a yeast one-hybrid library of A. oxytropis UA003 strain was constructed, and the transcriptional regulatory factors that may bind to the high-transcriptional activity region of the upstream non-coding region of the swnK gene were screened by this system. The results showed that the high transcriptional activity region was located at −656 bp and −392 bp of the upstream regulatory region of the swnK gene. A total of nine candidate transcriptional regulator molecules, including a C2H2 type transcription factor, seven annotated proteins, and an unannotated protein, were screened out through the Y1H system, which were bound to the upstream high transcriptional activity region of the swnK gene. This study provides new insight into the transcriptional regulation of the swnK gene and lays the foundation for further exploration of the regulatory mechanisms of SW biosynthesis in fungal endophytic locoweeds.

Abstract Background Xylan is one of the most abundant hemicelluloses and can crosslink cellulose and lignin to increase the stability of cell walls. A number of genes encoding glycosyltransferases play vital roles in xylan biosynthesis in plants, such as those of the GT43 family. However, little is known about glycosyltransferases in bamboo, especially woody bamboo which is a good substitute for timber. Results A total of 17 GT43 genes (PeGT43–1 ~ PeGT43–17) were identified in the genome of moso bamboo (Phyllostachys edulis), which belong to three subfamilies with specific motifs. The phylogenetic and collinearity analyses showed that PeGT43s may have undergone gene duplication, as a result of collinearity found in 12 pairs of PeGT43s, and between 17 PeGT43s and 10 OsGT43s. A set of cis-acting elements such as hormones, abiotic stress response and MYB binding elements were found in the promoter of PeGT43s. PeGT43s were expressed differently in 26 tissues, among which the highest expression level was found in the shoots, especially in the rapid elongation zone and nodes. The genes coexpressed with PeGT43s were annotated as associated with polysaccharide metabolism and cell wall biosynthesis. qRT–PCR results showed that the coexpressed genes had similar expression patterns with a significant increase in 4.0 m shoots and a peak in 6.0 m shoots during fast growth. In addition, the xylan content and structural polysaccharide staining intensity in bamboo shoots showed a strong positive correlation with the expression of PeGT43s. Yeast one-hybrid assays demonstrated that PeMYB35 could recognize the 5′ UTR/promoter of PeGT43–5 by binding to the SMRE cis-elements. Conclusions PeGT43s were found to be adapted to the requirement of xylan biosynthesis during rapid cell elongation and cell wall accumulation, as evidenced by the expression profile of PeGT43s and the rate of xylan accumulation in bamboo shoots. Yeast one-hybrid analysis suggested that PeMYB35 might be involved in xylan biosynthesis by regulating the expression of PeGT43–5 by binding to its 5′ UTR/promoter. Our study provides a comprehensive understanding of PeGT43s in moso bamboo and lays a foundation for further functional analysis of PeGT43s for xylan biosynthesis during rapid growth.

To screen the upstream regulatory transcription factors of cembratrienol synthase（NtCBT）in the cembranoids synthesis pathway of tobacco（Nicotiana tabacum L.），the promoter of NtCBT gene was cut into P1 to P6 regions，six yeast one-hybrid bait vectors pAbAi-Px were constructed and transformed into Y1H competent yeast cells，bait strains were constructed，and a self-activation experiment was completed. Furthermore，a screening was finished from yeast cDNA library of tobacco trichomes in order to obtain the transcription factors that interacted with P5 regions（-279 - -119 bp）. The results showed that，the growths of the strains containing P1 to P5 regions bait strain were inhibited on the medium added with 200 ng/mL AbA. A total of 49 positive colonies were obtained through Y1H screening when P5 regions as bait strain，among them，35 colonies were non-repeating sequences，and 3 of colonies were annotated as transcription factors of ANL2， ML1 and NF-Y. Above results lay a foundation for further study of gene expression and regulation mechanism of NtCBT. [ABSTRACT FROM AUTHOR]

Abstract Background Yeast one-hybrid (Y1H) is a common technique for identifying DNA-protein interactions, and robotic platforms have been developed for high-throughput analyses to unravel the gene regulatory networks in many organisms. Use of these high-throughput techniques has led to the generation of increasingly large datasets, and several software packages have been developed to analyze such data. We previously established the currently most efficient Y1H system, meiosis-directed Y1H; however, the available software tools were not designed for processing the additional parameters suggested by meiosis-directed Y1H to avoid false positives and required programming skills for operation. Results We developed a new tool named GateMultiplex with high computing performance using C++. GateMultiplex incorporated a graphical user interface (GUI), which allows the operation without any programming skills. Flexible parameter options were designed for multiple experimental purposes to enable the application of GateMultiplex even beyond Y1H platforms. We further demonstrated the data analysis from other three fields using GateMultiplex, the identification of lead compounds in preclinical cancer drug discovery, the crop line selection in precision agriculture, and the ocean pollution detection from deep-sea fishery. Conclusions The user-friendly GUI, fast C++ computing speed, flexible parameter setting, and applicability of GateMultiplex facilitate the feasibility of large-scale data analysis in life science fields.

BRANCHED1 (BRC1) is a crucial member of the TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) gene family and is well known for playing a central role in shoot branching by controlling buds’ paradormancy. However, the expression characteristics and molecular regulatory mechanism of BRC1 during blueberry bud dormancy are unclear. To shed light on these topics, shoots of three blueberry cultivars with different chilling requirements (CRs) were decapitated in summer to induce paradormancy release and subjected to different levels of chilling in winter to induce endodormancy release. The results showed that the high-CR cultivar ‘Chandler’ had the strongest apical dominance among the three cultivars; additionally, the expression of VcTCP18, which is homologous to BRC1, was the highest under both the decapitation treatment and low-temperature treatment. The ‘Emerald’ cultivar, with a low CR, demonstrated the opposite trend. These findings suggest that VcTCP18 plays a negative regulatory role in bud break and that there may be a correlation between the CR and tree shape. Through yeast 1-hybrid (Y1H) assays, we finally screened 21 upstream regulatory genes, including eight transcription factors: zinc-finger homeodomain protein 1/4/5/9, MYB4, AP2-like ethylene-responsive transcription factor AINTEGUMENTA (ANT), ASIL2-like, and bHLH035. It was found that these upstream regulatory genes positively or negatively regulated the expression of VcTCP18 based on the transcriptome expression profile. In summary, this study enriched our understanding of the regulatory network of BRCl during bud dormancy and provided new insights into the function of BRC1.

Key message: The overexpression of HaCYC2c and its regulation on HaNDUA2 through transcriptional recognition are important for regulating the heteromorphous development and functional differentiation of ray and disc florets in sunflower. Flower symmetry is closely related to pollinator recruitment and individual fecundity for higher plants and is the main feature used to identify flower type in angiosperms. In sunflower, HaCYC2c regulates floral organ development and floral symmetry, but the specific detail remains unclear. In this study, sunflower long petal mutant (lpm) with HaCYC2c insertion mutation was used to investigate the regulating role of HaCYC2c in the morphogenesis of florets and the transformation of floral symmetry through phenotype, transcriptome, qRT-PCR, and possible protein-gene interactions analyses. Results showed that HaCYC2c was overexpressed after an insertion into the promoter region. This gene could recognize the cis-acting element GGTCCC in the promoter region of HaNDUA2 that might regulate HaNDUA2 and affect other related genes. As a consequence, the abnormal elongation of disc petals and the degradation of male reproductive system occurred at the early development of floral organ in sunflower. Furthermore, this insertion mutation resulted in floral symmetry transformation, from actinomorphy to zygomorphy, thereby making the tubular disc florets transformed into ray-like disc florets in sunflower lpm. The findings suggested that the overexpression of HaCYC2c and its control of HaNDUA2 through transcriptional recognition might be an important regulating node of the heteromorphous development and functional differentiation for ray and disc florets in sunflower. This node contributes to the understanding of the balance between pollinator recruitment capacity of ray florets and fertility of disc florets for the optimization of reproductive efficiency and enhancement of species competitiveness in sunflower. [ABSTRACT FROM AUTHOR]

APETALA2/ethylene responsive factors (AP2/ERF) are unique regulators in the plant kingdom and are involved in the whole life activity processes such as development, ripening, and biotic and abiotic stresses. In tomato (Solanum lycopersicum), there are 140 AP2/ERF genes; however, their functionality remains poorly understood. In this work, the 14th and 19th amino acid differences in the AP2 domain were used to distinguish DREB and ERF subfamily members. Even when the AP2 domain of 68 ERF proteins from 20 plant species and motifs in tomato DREB and ERF proteins were compared, the binding ability of DREB and ERF proteins with DRE/CRT and/or GCC boxes remained unknown. During fruit development and ripening, the expressions of 13 DREB and 19 ERF subfamily genes showed some regular changes, and the promoters of most genes had ARF, DRE/CRT, and/or GCC boxes. This suggests that these genes directly or indirectly respond to IAA and/or ethylene (ET) signals during fruit development and ripening. Moreover, some of these may feedback regulate IAA or ET biosynthesis. In addition, 16 EAR motif-containing ERF genes in tomato were expressed in many organs and their total transcripts per million (TPM) values exceeded those of other ERF genes in most organs. To determine whether the EAR motif in EAR motif-containing ERF proteins has repression function, their EAR motifs were retained or deleted in a yeast one-hybrid (YIH) assay. The results indicate that most of EAR motif-containing ERF proteins lost repression activity after deleting the EAR motif. Moreover, some of these were expressed during ripening. Thus, these EAR motif-containing ERF proteins play vital roles in balancing the regulatory functions of other ERF proteins by completing the DRE/CRT and/or GCC box sites of target genes to ensure normal growth and development in tomato. [ABSTRACT FROM AUTHOR]

Brown cotton fiber (BCF) is a unique raw material of naturally colored cotton (NCC). But characteristics of the regulatory gene network and metabolic components related to the proanthocyanidins biosynthesis pathway at various stages of its fiber development remain unclear. Here, the dynamic changes in proanthocyanidins biosynthesis components and transcripts in the BCF variety "Zong 1-61" and its white near-isogenic lines (NILs) "RT" were characterized at five fiber developmental stages (0, 5, 10, 15, and 20 days post-anthesis; DPA). Enrichment analysis of differentially expressed genes (DEGs), comparison of metabolome differences, and pathway enrichment analysis of a weighted gene correlation network analysis together revealed the dominant gene expression of flavonoid biosynthesis (FB), phenylpropanoid metabolisms, and some carbohydrate metabolisms at 15 or 20 DPA than white cotton. Eventually, 63 genes were identified from five modules putatively related to FB. Three R2R3-MYB and two bHLH transcription factors were predicted as the core genes. Further, GhANS , GhANR1 , and GhUFGT2 were preliminarily regulated by GhMYB46 , GhMYB6 , and GhMYB3 , respectively, according to yeast one-hybrid assays in vitro. Our findings provide an important transcriptional regulatory network of proanthocyanidins biosynthesis pathway and dynamic flavonoid metabolism profiles. [ABSTRACT FROM AUTHOR]

Brown cotton fiber (BCF) is a unique raw material of naturally colored cotton (NCC). But characteristics of the regulatory gene network and metabolic components related to the proanthocyanidins biosynthesis pathway at various stages of its fiber development remain unclear. Here, the dynamic changes in proanthocyanidins biosynthesis components and transcripts in the BCF variety “Zong 1-61” and its white near-isogenic lines (NILs) “RT” were characterized at five fiber developmental stages (0, 5, 10, 15, and 20 days post-anthesis; DPA). Enrichment analysis of differentially expressed genes (DEGs), comparison of metabolome differences, and pathway enrichment analysis of a weighted gene correlation network analysis together revealed the dominant gene expression of flavonoid biosynthesis (FB), phenylpropanoid metabolisms, and some carbohydrate metabolisms at 15 or 20 DPA than white cotton. Eventually, 63 genes were identified from five modules putatively related to FB. Three R2R3-MYB and two bHLH transcription factors were predicted as the core genes. Further, GhANS, GhANR1, and GhUFGT2 were preliminarily regulated by GhMYB46, GhMYB6, and GhMYB3, respectively, according to yeast one-hybrid assays in vitro. Our findings provide an important transcriptional regulatory network of proanthocyanidins biosynthesis pathway and dynamic flavonoid metabolism profiles.

Background: Xylan is one of the most abundant hemicelluloses and can crosslink cellulose and lignin to increase the stability of cell walls. A number of genes encoding glycosyltransferases play vital roles in xylan biosynthesis in plants, such as those of the GT43 family. However, little is known about glycosyltransferases in bamboo, especially woody bamboo which is a good substitute for timber. Results: A total of 17 GT43 genes (PeGT43–1 ~ PeGT43–17) were identified in the genome of moso bamboo (Phyllostachys edulis), which belong to three subfamilies with specific motifs. The phylogenetic and collinearity analyses showed that PeGT43s may have undergone gene duplication, as a result of collinearity found in 12 pairs of PeGT43s, and between 17 PeGT43s and 10 OsGT43s. A set of cis-acting elements such as hormones, abiotic stress response and MYB binding elements were found in the promoter of PeGT43s. PeGT43s were expressed differently in 26 tissues, among which the highest expression level was found in the shoots, especially in the rapid elongation zone and nodes. The genes coexpressed with PeGT43s were annotated as associated with polysaccharide metabolism and cell wall biosynthesis. qRT–PCR results showed that the coexpressed genes had similar expression patterns with a significant increase in 4.0 m shoots and a peak in 6.0 m shoots during fast growth. In addition, the xylan content and structural polysaccharide staining intensity in bamboo shoots showed a strong positive correlation with the expression of PeGT43s. Yeast one-hybrid assays demonstrated that PeMYB35 could recognize the 5′ UTR/promoter of PeGT43–5 by binding to the SMRE cis-elements. Conclusions: PeGT43s were found to be adapted to the requirement of xylan biosynthesis during rapid cell elongation and cell wall accumulation, as evidenced by the expression profile of PeGT43s and the rate of xylan accumulation in bamboo shoots. Yeast one-hybrid analysis suggested that PeMYB35 might be involved in xylan biosynthesis by regulating the expression of PeGT43–5 by binding to its 5′ UTR/promoter. Our study provides a comprehensive understanding of PeGT43s in moso bamboo and lays a foundation for further functional analysis of PeGT43s for xylan biosynthesis during rapid growth. [ABSTRACT FROM AUTHOR]

Copyright of Acta Agronomica Sinica is the property of Crop Science Society of China and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Abstract Background ADP-glucose pyrophosphorylase (AGPase), the key enzyme in plant starch biosynthesis, is a heterotetramer composed of two identical large subunits and two identical small subunits. AGPase has plastidial and cytosolic isoforms in higher plants, whereas it is mainly detected in the cytosol of grain endosperms in cereal crops. Our previous results have shown that the expression of the TaAGPL1 gene, encoding the cytosolic large subunit of wheat AGPase, temporally coincides with the rate of starch accumulation and that its overexpression dramatically increases wheat AGPase activity and the rate of starch accumulation, suggesting an important role. Methods In this study, we performed yeast one-hybrid screening using the promoter of the TaAGPL1 gene as bait and a wheat grain cDNA library as prey to screen out the upstream regulators of TaAGPL1 gene. And the barley stripe mosaic virus-induced gene-silencing (BSMV-VIGS) method was used to verify the functional characterization of the identified regulators in starch biosynthesis. Results Disulfide isomerase 1-2 protein (TaPDIL1-2) was screened out, and its binding to the TaAGPL1-1D promoter was further verified using another yeast one-hybrid screen. Transiently silenced wheat plants of the TaPDIL1-2 gene were obtained by using BSMV-VIGS method under field conditions. In grains of BSMV-VIGS-TaPDIL1-2-silenced wheat plants, the TaAGPL1 gene transcription levels, grain starch contents, and 1000-kernel weight also significantly increased. Conclusions As important chaperones involved in oxidative protein folding, PDIL proteins have been reported to form hetero-dimers with some transcription factors, and thus, our results suggested that TaPDIL1-2 protein could indirectly and negatively regulate the expression of the TaAGPL1 gene and function in starch biosynthesis.

Background: Yeast one-hybrid (Y1H) is a common technique for identifying DNA-protein interactions, and robotic platforms have been developed for high-throughput analyses to unravel the gene regulatory networks in many organisms. Use of these high-throughput techniques has led to the generation of increasingly large datasets, and several software packages have been developed to analyze such data. We previously established the currently most efficient Y1H system, meiosis-directed Y1H; however, the available software tools were not designed for processing the additional parameters suggested by meiosis-directed Y1H to avoid false positives and required programming skills for operation. Results: We developed a new tool named GateMultiplex with high computing performance using C++. GateMultiplex incorporated a graphical user interface (GUI), which allows the operation without any programming skills. Flexible parameter options were designed for multiple experimental purposes to enable the application of GateMultiplex even beyond Y1H platforms. We further demonstrated the data analysis from other three fields using GateMultiplex, the identification of lead compounds in preclinical cancer drug discovery, the crop line selection in precision agriculture, and the ocean pollution detection from deep-sea fishery. Conclusions: The user-friendly GUI, fast C++ computing speed, flexible parameter setting, and applicability of GateMultiplex facilitate the feasibility of large-scale data analysis in life science fields. [ABSTRACT FROM AUTHOR]

The molecular networks involved in the regulation of HIV replication, transcription, and latency remain incompletely defined. To expand our understanding of these networks, we performed an unbiased high-throughput yeast one-hybrid screen, which identified 42 human transcription factors and 85 total protein-DNA interactions with HIV-1 and HIV-2 long terminal repeats. We investigated a subset of these transcription factors for transcriptional activity in cell-based models of infection. KLF2 and KLF3 repressed HIV-1 and HIV-2 transcription in CD4+ T cells, whereas PLAGL1 activated transcription of HIV-2 through direct protein-DNA interactions. Using computational modeling with interacting proteins, we leveraged the results from our screen to identify putative pathways that define intrinsic transcriptional networks. Overall, we used a high-throughput functional screen, computational modeling, and biochemical assays to identify and confirm several candidate transcription factors and biochemical processes that influence HIV-1 and HIV-2 transcription and latency. [ABSTRACT FROM AUTHOR]

Summary: Adverse environmental conditions reduce crop productivity and often increase the load of unfolded or misfolded proteins in the endoplasmic reticulum (ER). This potentially lethal condition, known as ER stress, is buffered by the unfolded protein response (UPR), a set of signaling pathways designed to either recover ER functionality or ignite programmed cell death. Despite the biological significance of the UPR to the life of the organism, the regulatory transcriptional landscape underpinning ER stress management is largely unmapped, especially in crops. To fill this significant knowledge gap, we performed a large‐scale systems‐level analysis of the protein–DNA interaction (PDI) network in maize (Zea mays). Using 23 promoter fragments of six UPR marker genes in a high‐throughput enhanced yeast one‐hybrid assay, we identified a highly interconnected network of 262 transcription factors (TFs) associated with significant biological traits and 831 PDIs underlying the UPR. We established a temporal hierarchy of TF binding to gene promoters within the same family as well as across different families of TFs. Cistrome analysis revealed the dynamic activities of a variety of cis‐regulatory elements (CREs) in ER stress‐responsive gene promoters. By integrating the cistrome results into a TF network analysis, we mapped a subnetwork of TFs associated with a CRE that may contribute to UPR management. Finally, we validated the role of a predicted network hub gene using the Arabidopsis system. The PDIs, TF networks, and CREs identified in our work are foundational resources for understanding transcription‐regulatory mechanisms in the stress responses and crop improvement. Significance Statement: Stress‐induced perturbations in the plant endoplasmic reticulum can result in (i) the accumulation of unfolded or misfolded proteins and (ii) crop loss. The unfolded protein response (UPR) functions to restore ER homeostasis but the gene products contributing to crops' UPR are largely unknown. To address this, we combined a high‐throughput enhanced yeast one‐hybrid assay with advanced genomics, and identified transcription factor networks and dynamic activities of cis‐regulatory elements controlling the activity of UPR genes in maize. [ABSTRACT FROM AUTHOR]

The Chinese white wax scale insect (CWWSI), Ericerus pela, can secret an amount of wax equivalent to their body weight. Previous studies demonstrated the fatty acyl-CoA reductase (far3) plays a pivotal role in wax secretion of CWWSI. The high expression of far3 is crucial for the massive wax secretion. However, the transcription regulation of far3 was not clear. To identify regulatory factors that control the expression of far3, the assay for transposase-accessible chromatin (ATAC) and yeast one-hybrid (Y1H) were carried out in this study. The ATAC sequencing of the CWWSI at the early wax-secretion stage ATAC-seq resulted in 22.75 GB raw data, generated 75,827,225 clean reads and revealed 142,771 peaks. There was one significant peak in the 3 kb upstream regulation regions. The peak sequence is located between -1000 and -670 bp upstream of the far3 transcription start site, spanning a length of 331 bp. This peak sequence served as bait for creating the pAbAi-peak recombinant vector, used in Y1H screenings to identify proteins interacting with far3 gene. The results indicate a successful CWWSI cDNA library construction with a capacity of 1.2 × 10 7 colony forming unit, a 95.8% recombination rate, and insert sizes between 1,000 and 2,000 bp. Self-activation tests established that 100 ng/mL of AbA effectively inhibited bait vector self-activation. Finally, a total of 88 positive clones were selected. After sequencing and removal of duplication, 63 unique clones were obtained from these screened colonies. By aligning the clone sequences with full-length transcriptome and genome of CWWSI, the full-length coding sequences of these clones were obtained. BlastX analysis identified a transcription factor, nuclear transcription factor Y beta, and two co-activators, cAMP-response-element-binding-protein-binding protein and WW domain binding protein 2. Reverse transcription quantitative polymerase chain reaction analysis confirmed that their expression patterns were consistent with the developmental stages preceding wax secretion and matched the wax secretion characteristics during ovulation periods. These results are beneficial for further research into the regulatory mechanisms of wax secretion of CWWSI., (© 2024 Wiley Periodicals LLC.)

Knotted1-like Homeobox (KNOX) proteins play important roles in regulating plant growth, development, and other biological processes. However, little information is available on the KNOX gene family in apple (Malus domestica Borkh.). In this study, 22 KNOX genes were identified in the apple genome. The gene structure, protein characteristics, and promoter region were characterized. The MdKNOX family members were divided into three classes based on their phylogenetic relationships. Quantitative real-time PCR analysis revealed that the majority of MdKNOX genes exhibited strongly preferential expression in buds and were significantly up-regulated during the flower induction period. The transcript levels of MdKNOX genes were responsive to treatments with flowering- and stress-related hormones. The putative upstream regulation factor MdGRF could directly bind to the promoter of MdKNOX15 and MdKNOX19, and inhibit their transcriptional activities, which were confirmed by yeast one-hybrid and dual-luciferase assays. The results provide an important foundation for future analysis of the regulation and functions of the MdKNOX gene family.

Knotted1-like Homeobox (KNOX) proteins play important roles in regulating plant growth, development, and other biological processes. However, little information is available on the KNOX gene family in apple (Malus domestica Borkh.). In this study, 22 KNOX genes were identified in the apple genome. The gene structure, protein characteristics, and promoter region were characterized. The MdKNOX family members were divided into three classes based on their phylogenetic relationships. Quantitative real-time PCR analysis revealed that the majority of MdKNOX genes exhibited strongly preferential expression in buds and were significantly up-regulated during the flower induction period. The transcript levels of MdKNOX genes were responsive to treatments with flowering- and stress-related hormones. The putative upstream regulation factor MdGRF could directly bind to the promoter of MdKNOX15 and MdKNOX19 , and inhibit their transcriptional activities, which were confirmed by yeast one-hybrid and dual-luciferase assays. The results provide an important foundation for future analysis of the regulation and functions of the MdKNOX gene family. [ABSTRACT FROM AUTHOR]

Soybean homeobox gene GmSBH1 has previously been proven to be involved in response to high temperature and humidity (HTH) stress. To investigate its expression patterns and active cis-elements, a 2040-bp 5’-upstream genomic DNA fragment of GmSBH1, named GmSBH1P, was isolated by PCR walking and characterized. Sequence analysis revealed that the fragment contains a series of cis-acting elements related to stress responses. The transient expression assay in the leaves of Nicotiana benthamiana and in the cotyledonary nodes of soybean indicated that the GmSBH1P strongly and rapidly mediates the induction of GUS expression under HTH stress. Deletion and mutation analysis of the promoter indicated that the cis-acting HSE (GAACTTTC) in GmSBH1P is essential for the promoter in response to HTH stress. In addition, 82 different proteins were identified to bind the cis-element by a yeast onehybrid system. These results indicated that the cloned promoter GmSBH1P could be applied to enhance the resistance to HTH stress, and the HSE would be an ideal candidate for mediating the expression of HTH-responsive genes in plants [ABSTRACT FROM AUTHOR]

basic-region helix-loop-helix (bHLH) proteins are a superfamily of transcription factors that are often involved in the control of growth and differentiation. Recently, it has been reported that the bHLH transcription factor DevR is involved in both asexual and sexual development in A. nidulans and regulates the conidial melanin production in A. fumigatus. In this study, we identified and characterized an A. oryzae gene, which showed high similarity with devR of A. nidulans and A. fumigatus. In the AodevR-disrupted strain, growth was delayed and the number of conidia was decreased on CD minimal agar plates, but the conidiation could be partially recovered by adding 0.6 M KCl. Simultaneously, the overexpression of AodevR was induced and resulted in extremely poor growth when the carbon source changed from glucose to polysaccharide (dextrin) in the CD agar plate. SEM indicated that the overexpression of AodevR resulted in extremely thin aberrant hyphal morphology. Conversely, the deletion of AodevR resulted in thicker hyphae and resulted in more resistance to Congo red relative to the control strain. Quantitative RT-PCR further indicated that AoDevR significantly affects chitin and starch metabolism, and especially, the overexpression of AodevR importantly inhibited the expression of the genes related to starch degradation. Yeast one-hybrid assay suggested that the DevR protein possibly interacted with promoter of amyR, which encodes a transcription factor involved in amylase production. In concert, AoDevR is importantly involved in polysaccharide metabolism and affects the growth of the A. oryzae strain. [ABSTRACT FROM AUTHOR]

Coptis chinensis Franch is a perennial species with high medical value. The rhizome of C. chinensis is a traditional Chinese medicine widely used for more than 2000 years in China. Its principal active ingredients are benzylisoquinoline alkaloids (BIAs). The basic helix-loop-helix (bHLH) transcription factors play an important regulatory role in the biosynthesis of plant secondary metabolites. However, the bHLH genes in C. chinensis have not been described, and little is known about their roles in alkaloid biosynthesis. In this study, a total of 143 Cc bHLH genes (Cc bHLHs) were identified and unevenly distributed on nine chromosomes. Phylogenetic analysis divided the 143 CcbHLH proteins into 26 subfamilies by comparison with Arabidopsis thaliana bHLH proteins. The majority CcbHLHs in each subgroup had similar gene structures and conserved motifs. Furthermore, the physicochemical properties, conserved motif, intron/exon composition, and cis-acting elements of CcbHLHs were analyzed. Transcriptome analysis revealed that 30 CcbHLHs were significantly expressed in the rhizomes of C. chinensis. Co-expression analysis revealed that 11 CcbHLHs were highly positively correlated with contents of various alkaloids of C. chinensis. Moreover, yeast one-hybrid experiments verified that CcbHLH001 and CcbHLH0002 could interact with the promoters of berberine biosynthesis pathway genes CcBBE and CcCAS , suggesting their regulatory roles in BIA biosynthesis. This study provides comprehensive insights into the bHLH gene family in C. chinensis and will support in-depth functional characterization of CcbHLHs involved in the regulation of protoberberine-type alkaloid biosynthesis. • A total of 143 CcbHLHs were divided into 26 subfamilies. • 11 CcbHLHs were highly positively correlated with contents of BIAs of C. chinensis by co-expression analysis and verified by qPCR analysis. • Y1H verified that CcbHLH001 and CcbHLH0002 interacted with the promoters of berberine biosynthesis pathway genes CcBBE and CcCAS. • This study suggests the regulatory roles of bHLH transcription factor in BIA biosynthesis and provides reference for further study on the regulation mechanism of BIAs biosynthesis in C. chinensis. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Southern Agriculture is the property of Journal of Southern Agriculture and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

【Objective】As one of the three TFL genes in grapevine, VlTFL1 A has been reported to play important roles in the formation of inflorescence. To provides a basis for further study of its expression and regulation, regulators that interact with the the promoter region of VlTFL1 A were screened. 【Method】A bait vector, which was constructed by the promoter region of VlTFL1 A, was transformed yeast cells to build bait yeast strain. The yeast one-hybrid c DNA library was constructed via SMART technology. Then the c DNA library and p GADT7-Rec vector was co-tranformed into the bait yeast strain to screen upstream regulators of the promoter region of VlTFL1 A via homologous recombination. 【Result】Nine positive yeast colonies that greater than 500 bp were screened out, and sequencing analysis and Blast homology analysis revealed that regulators that acted at the promoter region of VlTFL1 A were signal recognition particle receptor subunit alpha homolog, calmodulin-related protein, transketolase, xyloglucan glycosyltransferase 6, F-box protein At2 g32560-like, coronatine insensitive 1(COI1), UVB-induced protein At3 g17800, heavy metal-associated isoprenylated plant protein 39, and one uncharacterized protein LOC100254834.【Conclusion】Nine upstream regulators of the promoter region of VlTFL1 A were sreened out by yeast one-hybrid technology, but their functions in inflorescence formaion of grapevine has not been reported. The result provided candidate regulators for further study of VlTFL1 A expression and regulation. [ABSTRACT FROM AUTHOR]

The Auxin/ Indole acetic acid ( Aux/ IAA) genes are auxin primary response genes that play a pivotal role in apical dominance, phototropism, and embryonic development. In this study, we cloned the promoter of BpIAA10 and constructed a BpProIAA::Luc vector for transformation into Arabidopsis thaliana. The results indicated that BpIAA10 is expressed in the apical meristem, embryonic leaves, and young stem. The tissue-specific expression pattern was same in A. thaliana and Betula platyphylla. Additionally, IAA, IBA, GA, SA, and ABA could induce the expression of BpIAA10 while MeJA inhibited the expression of BpIAA10, suggesting that plant hormones influence the expression of BpIAA10. Strong light conditions also markedly induced the expression of BpIAA10, indicating that BpIAA10 is sensitive to light intensity and that light may be sufficient but not necessary for the normal expression of BpIAA10. Conversely, IBA-induced expression of BpIAA10 was suppressed with strong light, suggesting that IBA-induced BpIAA10 expression is partly light dependent. We predicted the presence of cis-acting elements in the BpIAA10 promoter and found many different types cis-acting elements. Furthermore, promoter analysis and yeast one-hybrid assay revealed that the bait2 (TGA-element) can specifically bind to transcription factors 14-3-3 and Ribosomal_L2, and the bait3 (ntBBF1ARROLB) can specifically bind to FAR1, 14-3-3 Like and kinesin-like protein. Therefore, our study characterized the expression of BpIAA10 and found five transcription factors that regulate expression of BpIAA10. This provides a basis for determining the function of BpIAA10. [ABSTRACT FROM AUTHOR]

Heavy metal pollution of soil has become a serious global concern in recent years. In particular, cadmium (Cd) contamination is a ubiquitous environmental problem among heavy metals. Iris lactea var. chinensis is a perennial herb with significant ecological and industrial value. It is known for its good cadmium tolerance and cadmium enrichment capabilities; thus, it is considered an important plant for the restoration of cadmium-contaminated soil. Previous studies have shown that the cysteine-rich gene, IlCDT1 isolated from I. lactea conferred tolerance to Cd stress in yeast and Arabidopsis thaliana. However, the molecular mechanism of its regulation is poorly understood to date. In the current study, we screened and identified the transcription factor IlNF-YC6 that regulates IlCDT1 using yeast one-hybrid assay (Y1H). Overexpression of IlNF-YC6 conferred a significant tolerance to Cd stress in A. thaliana. Furthermore, the detection of physiological markers and transcriptome analysis revealed that IlNF-YC6 might increase plant tolerance to Cd stress primarily by improving plant detoxification ability and antioxidant capacity. Overall, our study would provide a genetic resource for candidate gene selection that might enhance plant tolerance to Cd contamination in soil or groundwater and subsequently lowering Cd absorption in the future. [Display omitted] • Upstream regulators IlNF-YC6 of IlCDT1 identified by yeast one-hybrid. • IlNF-YC6 is up-regulated by cadmium stress and interacts with CCAAT element. • Overexpression IlNF-YC6 in A. thaliana significantly increased cadmium tolerance. • RNA-seq data demonstrates that I1NF-YC6 can enhance cadmium tolerance in plants by activating IlCDT1 expression. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine how the mulberry (Morus notabilis) polyphenol oxidase 1 gene (MnPPO1) is regulated during plant stress responses by exploring the interaction between its promoter region and regulatory transcription factors. First, we analyzed the cis-acting elements in the MnPPO1 promoter. Then, we used the MnPPO1 promoter region [(1268 bp, including an MYB3R-binding cis-element (MSA)] as a probe to capture proteins in DNA pull-down assays. These analyses revealed that the MYB3R1 transcription factor in M. notabilis (encoded by MnMYB3R1) binds to the MnPPO1 promoter region. We further explored the interaction between the MnPPO1 promoter and MYB3R1 with the dual luciferase reporter, yeast one-hybrid, and chromatin immunoprecipitation assays. These analyses verified that MnMYB3R1 binds to the MSA in the MnPPO1 promoter region. The overexpression of MnMYB3R1 in tobacco upregulated the expression of the tobacco PPO gene. This observation as well as the quantitative real-time PCR results implied that MnMYB3R1 and PPO are involved in the abscisic acid-responsive stress response pathway.

WRKY transcription factors play important roles in many biological processes, and mainly bind to the W-box element to regulate gene expression. Previously, we characterized a WRKY gene from Tamarix hispida , ThWRKY4 , in response to abiotic stress, and showed that it bound to the W-box motif. However, whether ThWRKY4 could bind to other motifs remains unknown. In this study, we employed a Transcription Factor-Centered Yeast one Hybrid (TF-Centered Y1H) screen to study the motifs recognized by ThWRKY4. In addition to the W-box core cis -element (termed W-box), we identified that ThWRKY4 could bind to two other motifs: the RAV1A element (CAACA) and a novel motif with sequence of GTCTA (W-box like sequence, WLS). The distributions of these motifs were screened in the promoter regions of genes regulated by some WRKYs. The results showed that the W-box, RAV1A, and WLS motifs were all present in high numbers, suggesting that they play key roles in gene expression mediated by WRKYs. Furthermore, five WRKY proteins from different WRKY subfamilies in Arabidopsis thaliana were selected and confirmed to bind to the RAV1A and WLS motifs, indicating that they are recognized commonly by WRKYs. These findings will help to further reveal the functions of WRKY proteins. [ABSTRACT FROM AUTHOR]

The translation of the genotype into phenotype, represented for example by the expression of genes encoding enzymes required for the biosynthesis of phytochemicals that are important for interaction of plants with the environment, is largely carried out by transcription factors (TFs) that recognize specific cis -regulatory elements in the genes that they control. TFs and their target genes are organized in gene regulatory networks (GRNs), and thus uncovering GRN architecture presents an important biological challenge necessary to explain gene regulation. Linking TFs to the genes they control, central to understanding GRNs, can be carried out using gene- or TF-centered approaches. In this study, we employed a gene-centered approach utilizing the yeast one-hybrid assay to generate a network of protein–DNA interactions that participate in the transcriptional control of genes involved in the biosynthesis of maize phenolic compounds including general phenylpropanoids, lignins, and flavonoids. We identified 1100 protein–DNA interactions involving 54 phenolic gene promoters and 568 TFs. A set of 11 TFs recognized 10 or more promoters, suggesting a role in coordinating pathway gene expression. The integration of the gene-centered network with information derived from TF-centered approaches provides a foundation for a phenolics GRN characterized by interlaced feed-forward loops that link developmental regulators with biosynthetic genes. [ABSTRACT FROM AUTHOR]

Algae represent a unique opportunity to develop a sustainable production platform for many valuable products. A key hindrance to exploiting algae's potential as a bio-product manufacturing platform is our limited understanding of nuclear genetic regulatory systems. To begin to address this deficiency we identified prospective transcription factor-promoter pairs that have the potential for use in a controllable synthetic transcription system. We constructed a library of 92 recombinant nuclear transcription factors (TFs) from the model alga Chlamydomonas reinhardtii . Using a yeast one-hybrid assay, we identified TFs with the ability to activate transcription and chose TF64 for further analysis. We generated two independent C. reinhardtii strains that constitutively expressed the gene encoding TF64 and used RNA-sequencing on these strains to identify transcripts that were altered by expression of the TF's gene. Many mRNAs were regulated by TF64, and we confirmed activation of transcription of nine genes from the LHCBM family by RT-qPCR. Overall, we were able to identify numerous potential TF-cognate promoter binding partners in C. reinhardtii , thus laying the groundwork to develop a controllable synthetic nuclear transcription system for microalgae, an essential tool for realizing their full biotechnological potential. [ABSTRACT FROM AUTHOR]

NAC (NAM, ATAF1/2, and CUC2) transcription factors play important roles in many biological processes, and mainly bind to the NACRS with core sequences “CACG” or “CATGTG” to regulate gene expression. However, whether NAC proteins can bind to other motifs without these core sequences remains unknown. In this study, we employed a Transcription Factor-Centered Yeast one Hybrid (TF-Centered Y1H) screen to study the motifs recognized by ANAC074. In addition to the NACRS core cis-element, we identified that ANAC074 could bind to MybSt1, NRS1, and NRS2. Y1H and GUS assays showed that ANAC074 could bind the promoters of ethylene responsive genes and stress responsive genes via the NRS1, NRS2, or MybSt1 element. ChIP study further confirmed that the bindings of ANAC074 to MybSt1, NRS1, and NRS2 actually occurred in Arabidopsis. Furthermore, ten NAC proteins from different NAC subfamilies in Arabidopsis thaliana were selected and confirmed to bind to the MybSt1, NRS1, and NRS2 motifs, indicating that they are recognized commonly by NACs. These findings will help us to further reveal the functions of NAC proteins.

The transcription factors of basic leucine zipper (bZIP) family genes play significant roles in stress response as well as growth and development in plants. However, little is known about the bZIP gene family in Chinese chestnut ( Castanea mollissima Blume). To better understand the characteristics of bZIP s in chestnut and their function in starch accumulation, a series of analyses were performed including phylogenetic, synteny, co-expression and yeast one-hybrid analyses. Totally, we identified 59 bZIP genes that were unevenly distributed in the chestnut genome and named them CmbZIP01 to CmbZIP59 . These CmbZIP s were clustered into 13 clades with clade-specific motifs and structures. A synteny analysis revealed that segmental duplication was the major driving force of expansion of the CmbZIP gene family. A total of 41 CmbZIP genes had syntenic relationships with four other species. The results from the co-expression analyses indicated that seven CmbZIP s in three key modules may be important in regulating starch accumulation in chestnut seeds. Yeast one-hybrid assays showed that transcription factors CmbZIP13 and CmbZIP35 might participate in starch accumulation in the chestnut seed by binding to the promoters of CmISA2 and CmSBE1_2 , respectively. Our study provided basic information on CmbZIP genes, which can be utilized in future functional analysis and breeding studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zhang, Liu, Jia, Wang, Lu, Wang, Zhang, Zhang and Wang.)

Transcription factors play a key role to enable plants to cope with abiotic stresses. DREB2 regulates the expression of several stress-inducible genes and constitutes major hubs in the water stress signalling webs. We cloned and characterized a novel gene encoding the Fp DREB2A transcription factor from Fraxinus pennsylvanica, and a yeast activity assay confirmed its DRE binding and transcription activation. Overexpression of Fp DREB2A in R. pseudoacacia showed enhanced resistance to drought stress. The transgenic plant survival rate was significantly higher than that of WT in soil drying and re-watering treatments. Transgenic lines showed a dramatic change in root architecture, and horizontal and vertical roots were found in transgenic plants compared to WT. The vertical roots penetrated in the field soil to more than 60 cm deep, while horizontal roots expanded within the top 20-30 cm of the soil. A physiological test demonstrated that chlorophyll contents were more gradually reduced and that soluble sugars and proline levels elevated more sharply but malondialdehyde level stayed the same ( P < 0.05). Plant hormone levels of abscisic acid and IAA were higher than that of WT, while gibberellins and zeatin riboside were found to be lower. The root transcriptomes were sequenced and annotated into 2011 differential expression genes ( DEGs). The DEGs were categorized in 149 pathways and were found to be involved in plant hormone signalling, transcription factors, stimulus responses, phenylalanine, carbohydrate and other metabolic pathways. The modified pathways in plant hormone signalling are thought to be the main cause of greater horizontal and vertical root development, in particular. [ABSTRACT FROM AUTHOR]

Bud dormancy in perennial plants is indispensable to survival over winter and to regrowth and development in the following year. However, the molecular pathways of endo-dormancy induction, maintenance, and release are still unclear, especially in fruit crops. To identify genes with roles in regulating endo-dormancy, 30 MIKCC-type MADS-box genes were identified in the pear genome and characterized. The 30 genes were analysed to determine their phylogenetic relationships with homologous genes, genome locations, gene structure, tissue-specific transcript profiles, and transcriptional patterns during flower bud dormancy in 'Suli' pear (Pyrus pyrifolia white pear group). The roles in regulating bud dormancy varied among the MIKC gene family members. Yeast one-hybrid and transient assays showed that PpCBF enhanced PpDAM1 and PpDAM3 transcriptional activity during the induction of dormancy, probably by binding to the C-repeat/DRE binding site, while DAM proteins inhibited the transcriptional activity of PpFT2 during dormancy release. In the small RNA-seq analysis, 185 conserved, 24 less-conserved, and 32 pear-specific miRNAs with distinct expression patterns during bud dormancy were identified. Joint analyses of miRNAs and MIKC genes together with degradome data showed that miR6390 targeted PpDAM transcripts and degraded them to release PpFT2. Our data show that cross-talk among PpCBF, PpDAM, PpFT2, and miR6390 played important roles in regulating endo-dormancy. A model for the molecular mechanism of dormancy transition is proposed: short-term chilling in autumn activates the accumulation of CBF, which directly promotes DAM expression; DAM subsequently inhibits FT expression to induce endo-dormancy, and miR6390 degrades DAM genes to release endo-dormancy. [ABSTRACT FROM AUTHOR]

AP2/ERF is a large transcription factor family that regulates plant physiological processes, such as plant development and stress response. Carrot ( Daucus carota L.) is an important economical crop with a genome size of 480 Mb; the draft genome sequencing of this crop has been completed by our group. However, little is known about the AP2/ERF factors in carrot. In this study, a total of 267 putative AP2/ERF factors were identified from the whole-genome sequence of carrot. These AP2/ERF proteins were phylogenetically clustered into five subfamilies based on their similarity to the amino acid sequences from Arabidopsis. The distribution and comparative genome analysis of the AP2/ERF factors among plants showed the AP2/ERF factors had expansion during the evolutionary process, and the AP2 domain was highly conserved during evolution. The number of AP2/ERF factors in land plants expanded during their evolution. A total of 60 orthologous and 145 coorthologous AP2/ERF gene pairs between carrot and Arabidopsis were identified, and the interaction network of orthologous genes was constructed. The expression patterns of eight AP2/ERF family genes from each subfamily (DREB, ERF, AP2, and RAV) were related to abiotic stresses. Yeast one-hybrid and β-galactosidase activity assays confirmed the DRE and GCC box-binding activities of DREB subfamily genes. This study is the first to identify and characterize the AP2/ERF transcription factors in carrot using whole-genome analysis, and the findings may serve as references for future functional research on the transcription factors in carrot. [ABSTRACT FROM AUTHOR]

Flavonoid 3′-hydroxylase (F3′H), a member of cytochrome P450 protein family, introduces B-ring hydroxyl group in the 3′ position of the flavonoid. In this study, the cDNA sequence of a F3′H gene ( VviF3′H ), which contains an open reading frame of 1530 bp encoding a polypeptide of 509 amino acids, was cloned and characterized from Vitis vinifera L. cv. Cabernet Sauvignon. VviF3′H showed high homology to known F3′H genes, especially F3′Hs from the V. vinifera reference genome (Pinot Noir) and lotus. Expression profiling analysis using real-time PCR revealed that VviF3′H was ubiquitously expressed in all tested tissues including berries, leaves, flowers, roots, stems and tendrils, suggesting its important physiological role in plant growth and development. Moreover, the transcript level of VviF3′H gene in grape berries was relatively higher at early developmental stages and gradually decreased during véraison, and then increased in the mature phase. In addition, the promoter of VviF3′H was isolated by using TAIL-PCR. Yeast one-hybrid screening of the Cabernet Sauvignon cDNA library and subsequent in vivo / vitro validations revealed the interaction between VviF3′H promoter and several transcription factors, including members of HD-Zip, NAC, MYB and EIN families. A transcriptional regulation mechanism of VviF3′H expression is proposed for the first time. [ABSTRACT FROM AUTHOR]