DeepGRNCS: deep learning-based framework for jointly inferring gene regulatory networks across cell subpopulations.

Inferring gene regulatory networks (GRNs) allows us to obtain a deeper understanding of cellular function and disease pathogenesis. Recent advances in single-cell RNA sequencing (scRNA-seq) technology have improved the accuracy of GRN inference. However, many methods for inferring individual GRNs from scRNA-seq data are limited because they overlook intercellular heterogeneity and similarities between different cell subpopulations, which are often present in the data. Here, we propose a deep learning-based framework, DeepGRNCS, for jointly inferring GRNs across cell subpopulations. We follow the commonly accepted hypothesis that the expression of a target gene can be predicted based on the expression of transcription factors (TFs) due to underlying regulatory relationships. We initially processed scRNA-seq data by discretizing data scattering using the equal-width method. Then, we trained deep learning models to predict target gene expression from TFs. By individually removing each TF from the expression matrix, we used pre-trained deep model predictions to infer regulatory relationships between TFs and genes, thereby constructing the GRN. Our method outperforms existing GRN inference methods for various simulated and real scRNA-seq datasets. Finally, we applied DeepGRNCS to non-small cell lung cancer scRNA-seq data to identify key genes in each cell subpopulation and analyzed their biological relevance. In conclusion, DeepGRNCS effectively predicts cell subpopulation-specific GRNs. The source code is available at https://github.com/Nastume777/DeepGRNCS.

A fungal metabolic regulator underlies infectious synergism during Candida albicans-Staphylococcus aureus intra-abdominal co-infection.

Candida albicans and Staphylococcus aureus are two commonly associated pathogens that cause nosocomial infections with high morbidity and mortality. Our prior and current work using a murine model of polymicrobial intra-abdominal infection (IAI) demonstrates that synergistic lethality is driven by Candida-induced upregulation of functional S. aureus α-toxin leading to polymicrobial sepsis and organ damage. In order to determine the candidal effector(s) mediating enhanced virulence, an unbiased screen of C. albicans transcription factor mutants was undertaken revealing that zcf13Δ/Δ fails to drive augmented α-toxin or lethal synergism during co-infection. A combination of transcriptional and phenotypic profiling approaches shows that ZCF13 regulates genes involved in pentose metabolism, including RBK1 and HGT7 that contribute to fungal ribose catabolism and uptake, respectively. Subsequent experiments reveal that ribose inhibits the staphylococcal agr quorum sensing system and concomitantly represses toxicity. Unlike wild-type C. albicans, zcf13Δ/Δ did not effectively utilize ribose during co-culture or co-infection leading to exogenous ribose accumulation and agr repression. Forced expression of RBK1 and HGT7 in the zcf13Δ/Δ mutant fully restores pathogenicity during co-infection. Collectively, our results detail the interwoven complexities of cross-kingdom interactions and highlight how intermicrobial metabolism impacts polymicrobial disease pathogenesis with devastating consequences for the host.

Co-regulatory network analysis of the main secondary metabolite (SM) biosynthesis in Crocus sativus L.

Saffron (Crocus sativus L.) is being embraced as the most important medicinal plant and the commercial source of saffron spice. Despite the beneficial economic and medicinal properties of saffron, the regulatory mechanism of the correlation of TFs and genes related to the biosynthesis of the apocarotenoids pathway is less obvious. Realizing these regulatory hierarchies of gene expression networks related to secondary metabolites production events is the main challenge owing to the complex and extensive interactions between the genetic behaviors. Recently, high throughput expression data have been highly feasible for constructing co-regulation networks to reveal the regulated processes and identifying novel candidate hub genes in response to complex processes of the biosynthesis of secondary metabolites. Herein, we performed Weighted Gene Co-expression Network Analysis (WGCNA), a systems biology method, to identify 11 regulated modules and hub TFs related to secondary metabolites. Three specialized modules were found in the apocarotenoids pathway. Several hub TFs were identified in notable modules, including MADS, C2H2, ERF, bZIP, HD-ZIP, and zinc finger protein MYB and HB, which were potentially associated with apocarotenoid biosynthesis. Furthermore, the expression levels of six hub TFs and six co-regulated genes of apocarotenoids were validated with RT-qPCR. The results confirmed that hub TFs specially MADS, C2H2, and ERF had a high correlation (P < 0.05) and a positive effect on genes under their control in apocarotenoid biosynthesis (CCD2, GLT2, and ADH) among different C. sativus ecotypes in which the metabolite contents were assayed. Promoter analysis of the co-expressed genes of the modules involved in apocarotenoids biosynthesis pathway suggested that not only are the genes co-expressed, but also share common regulatory motifs specially related to hub TFs of each module and that they may describe their common regulation. The result can be used to engineer valuable secondary metabolites of C. sativus by manipulating the hub regulatory TFs.

Genome-wide analysis of MYB transcription factor family and AsMYB1R subfamily contribution to ROS homeostasis regulation in Avena sativa under PEG-induced drought stress.

BACKGROUND: The myeloblastosis (MYB) transcription factor (TF) family is one of the largest and most important TF families in plants, playing an important role in a life cycle and abiotic stress. RESULTS: In this study, 268 Avena sativa MYB (AsMYB) TFs from Avena sativa were identified and named according to their order of location on the chromosomes, respectively. Phylogenetic analysis of the AsMYB and Arabidopsis MYB proteins were performed to determine their homology, the AsMYB1R proteins were classified into 5 subgroups, and the AsMYB2R proteins were classified into 34 subgroups. The conserved domains and gene structure were highly conserved among the subgroups. Eight differentially expressed AsMYB genes were screened in the transcriptome of transcriptional data and validated through RT-qPCR. Three genes in AsMYB2R subgroup, which are related to the shortened growth period, stomatal closure, and nutrient and water transport by PEG-induced drought stress, were investigated in more details. The AsMYB1R subgroup genes LHY and REV 1, together with GST, regulate ROS homeostasis to ensure ROS signal transduction and scavenge excess ROS to avoid oxidative damage. CONCLUSION: The results of this study confirmed that the AsMYB TFs family is involved in the homeostatic regulation of ROS under drought stress. This lays the foundation for further investigating the involvement of the AsMYB TFs family in regulating A. sativa drought response mechanisms.

Genome-wide analysis of R2R3-MYB transcription factors in poplar and functional validation of PagMYB147 in defense against Melampsora magnusiana.

MAIN CONCLUSION: Transcription of PagMYB147 was induced in poplar infected by Melampsora magnusiana, and a decline in its expression levels increases the host's susceptibility, whereas its overexpression promotes resistance to rust disease. Poplars are valuable tree species with diverse industrial and silvicultural applications. The R2R3-MYB subfamily of transcription factors plays a crucial role in response to biotic stresses. However, the functional studies on poplar R2R3-MYB genes in resistance to leaf rust disease are still insufficient. We identified 191 putative R2R3-MYB genes in the Populus trichocarpa genome. A phylogenetic analysis grouped poplar R2R3-MYBs and Arabidopsis R2R3-MYBs into 33 subgroups. We detected 12 tandem duplication events and 148 segmental duplication events, with the latter likely being the main contributor to the expansion of poplar R2R3-MYB genes. The promoter regions of these genes contained numerous cis-acting regulatory elements associated with response to stress and phytohormones. Analyses of RNA-Seq data identified a multiple R2R3-MYB genes response to Melampsora magnusiana (Mmag). Among them, PagMYB147 was significantly up-regulated under Mmag inoculation, salicylic acid (SA) and methyl jasmonate (MeJA) treatment, and its encoded product was primarily localized to the cell nucleus. Silencing of PagMYB147 exacerbated the severity of Mmag infection, likely because of decreased reactive oxygen species (ROS) production and phenylalanine ammonia-lyase (PAL) enzyme activity, and up-regulation of genes related to ROS scavenging and down-regulation of genes related to PAL, SA and JA signaling pathway. In contrast, plants overexpressing PagMYB147 showed the opposite ROS accumulation, PAL enzyme activity, SA and JA-related gene expressions, and improved Mmag resistance. Our findings suggest that PagMYB147 acts as a positive regulatory factor, affecting resistance in poplar to Mmag by its involvement in the regulation of ROS homeostasis, SA and JA signaling pathway.

BET Bromodomain Inhibition Potentiates Ocular Melanoma Therapy by Inducing Cell Cycle Arrest.

Purpose: Ocular melanoma is a common primary malignant ocular tumor in adults with limited effective treatments. Epigenetic regulation plays an important role in tumor development. The switching/sucrose nonfermentation (SWI/SNF) chromatin remodeling complex and bromodomain and extraterminal domain family proteins are epigenetic regulators involved in several cancers. We aimed to screen a candidate small molecule inhibitor targeting these regulators and investigate its effect and mechanism in ocular melanoma. Methods: We observed phenotypes caused by knockdown of the corresponding gene and synergistic effects with BRD inhibitor treatment and SWI/SNF complex knockdown. The effect of JQ-1 on ocular melanoma cell cycle and apoptosis was analyzed with flow cytometry. Via RNA sequencing, we also explored the mechanism of BRD4. Results: The best tumor inhibitory effect was observed for the BRD4 inhibitor (JQ-1), although there were no statistically obvious changes in the shBRD4 and shBRD9 groups. Interestingly, the inhibitory effect of JQ-1 was decrease in the shBRD4 group. JQ-1 inhibits the growth of melanoma in various cell lines and in tumor-bearing mice. We found 17 of these 28 common differentially expressed genes were downregulated after MEL270 and MEL290 cells treated with JQ-1. Four of these 17 genes, TP53I11, SH2D5, SEMA5A, and MDGA1, were positively correlated with BRD4. In TCGA database, low expression of TP53I11, SH2D5, SEMA5A, and MDGA1 improved the overall survival rate of patients. Furthermore, the disease-free survival rate was increased in the groups with low expression of TP53I11, SH2D5, and SEMA5A. Conclusions: JQ-1 may act downstream of BRD4 and suppress ocular melanoma growth by inducing G1 cell cycle arrest.

Cellular zinc status alters chromatin accessibility and binding of p53 to DNA.

Zn2+ is an essential metal required by approximately 850 human transcription factors. How these proteins acquire their essential Zn2+ cofactor and whether they are sensitive to changes in the labile Zn2+ pool in cells remain open questions. Using ATAC-seq to profile regions of accessible chromatin coupled with transcription factor enrichment analysis, we examined how increases and decreases in the labile zinc pool affect chromatin accessibility and transcription factor enrichment. We found 685 transcription factor motifs were differentially enriched, corresponding to 507 unique transcription factors. The pattern of perturbation and the types of transcription factors were notably different at promoters versus intergenic regions, with zinc-finger transcription factors strongly enriched in intergenic regions in elevated Zn2+ To test whether ATAC-seq and transcription factor enrichment analysis predictions correlate with changes in transcription factor binding, we used ChIP-qPCR to profile six p53 binding sites. We found that for five of the six targets, p53 binding correlates with the local accessibility determined by ATAC-seq. These results demonstrate that changes in labile zinc alter chromatin accessibility and transcription factor binding to DNA.

Epigenetic alterations affecting hematopoietic regulatory networks as drivers of mixed myeloid/lymphoid leukemia.

Leukemias with ambiguous lineage comprise several loosely defined entities, often without a clear mechanistic basis. Here, we extensively profile the epigenome and transcriptome of a subgroup of such leukemias with CpG Island Methylator Phenotype. These leukemias exhibit comparable hybrid myeloid/lymphoid epigenetic landscapes, yet heterogeneous genetic alterations, suggesting they are defined by their shared epigenetic profile rather than common genetic lesions. Gene expression enrichment reveals similarity with early T-cell precursor acute lymphoblastic leukemia and a lymphoid progenitor cell of origin. In line with this, integration of differential DNA methylation and gene expression shows widespread silencing of myeloid transcription factors. Moreover, binding sites for hematopoietic transcription factors, including CEBPA, SPI1 and LEF1, are uniquely inaccessible in these leukemias. Hypermethylation also results in loss of CTCF binding, accompanied by changes in chromatin interactions involving key transcription factors. In conclusion, epigenetic dysregulation, and not genetic lesions, explains the mixed phenotype of this group of leukemias with ambiguous lineage. The data collected here constitute a useful and comprehensive epigenomic reference for subsequent studies of acute myeloid leukemias, T-cell acute lymphoblastic leukemias and mixed-phenotype leukemias.

The dynamic expression of YAP is essential for the development of male germ cells derived from human embryonic stem cells.

YAP plays a vital role in controlling growth and differentiation in various cell lineages. Although the expression of YAP in mice testicular and spermatogenic cells suggests its role in mammalian spermatogenesis, the role of YAP in the development of human male germ cells has not yet been determined. Using an in vitro model and a gene editing approach, we generated human spermatogonia stem cell-like cells (hSSLCs) from human embryonic stem cells (hESCs) and investigated the role of YAP in human spermatogenesis. The results showed that reducing YAP expression during the early stage of spermatogenic differentiation increased the number of PLZF+ hSSLCs and haploid spermatid-like cells. We also demonstrated that the up-regulation of YAP is essential for maintaining spermatogenic cell survival during the later stages of spermatogenic differentiation. The expression of YAP that deviates from this pattern results in a lower number of hSSLCs and an increased level of spermatogenic cell death. Taken together, our result demonstrates that the dynamic expression pattern of YAP is essential for human spermatogenesis. Modulating the level of YAP during human spermatogenesis could improve the production yield of male germ cells derived from hESCs, which could provide the optimization method for in vitro gametogenesis and gain insight into the application in the treatment of male infertility.

Comprehensive identification and expression analysis of FAR1/FHY3 genes under drought stress in maize (Zea mays L.).

Background: FAR1/FHY3 transcription factors are derived from transposase, which play important roles in light signal transduction, growth and development, and response to stress by regulating downstream gene expression. Although many FAR1/FHY3 members have been identified in various species, the FAR1/FHY3 genes in maize are not well characterized and their function in drought are unknown. Method: The FAR1/FHY3 family in the maize genome was identified using PlantTFDB, Pfam, Smart, and NCBI-CDD websites. In order to investigate the evolution and functions of FAR1 genes in maize, the information of protein sequences, chromosome localization, subcellular localization, conserved motifs, evolutionary relationships and tissue expression patterns were analyzed by bioinformatics, and the expression patterns under drought stress were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Results: A total of 24 ZmFAR members in maize genome, which can be divided into five subfamilies, with large differences in protein and gene structures among subfamilies. The promoter regions of ZmFARs contain abundant abiotic stress-responsive and hormone-respovensive cis-elements. Among them, drought-responsive cis-elements are quite abundant. ZmFARs were expressed in all tissues detected, but the expression level varies widely. The expression of ZmFARs were mostly down-regulated in primary roots, seminal roots, lateral roots, and mesocotyls under water deficit. Most ZmFARs were down-regulated in root after PEG-simulated drought stress. Conclusions: We performed a genome-wide and systematic identification of FAR1/FHY3 genes in maize. And most ZmFARs were down-regulated in root after drought stress. These results indicate that FAR1/FHY3 transcription factors have important roles in drought stress response, which can lay a foundation for further analysis of the functions of ZmFARs in response to drought stress.

Interference with sphingosine kinase-1 reduces the hydrogen peroxide-induced oxidative stress damage in melanocytes through four and a half LIM domains 2.

Vitiligo is featured by manifestation of white maculae and primarily results from oxidative stress. Sphingosine kinase-1 (SPHK1) participates in oxidative stress. This paper was devised to explore the role of SPHK1 in vitiligo and to disclose the mechanism. PIG1 cell viability was appraised utilizing cell counting kit-8 assay while Western blot detected SPHK1 and four and a half LIM domains 2 (FHL2). The transduction efficacy of small interfering RNA (siRNA)-SPHK1, siRNA-FHL2 and pcDNA3.1 plasmid overexpressing FHL2 (Ov-FHL2) was checked using Western blot. Flow cytometry detected cell apoptotisis. Western blot detected mitochondrial cytochrome c (Mit-Cyt-c) and cytosolic cytochrome c (Cyto-Cyt-c). Dichloro-dihydro-fluorescein diacetate (DCFH-DA) detected reactive oxygen species (ROS) activity while oxidative stress markers were evaluated using corresponding assay kits. SPHK1 expression was discovered to be increased in hydrogen peroxide (H2O2)-challenged PIG1 cells and SPHK1 interference alleviated H2O2-challenged viability damage, apoptosis, oxidative stress and FHL2 expression in PIG1 cells. FHL2 depletion could suppress viability damage, apoptosis and oxidative stress in H2O2-challenged PIG1 cells. Rescue experiments demonstrated that the suppressive impacts of SPHK1 deficiency on PIG1 cell viability, apoptosis and oxidative stress induced by H2O2 were offset by FHL2 overexpression. Collectively, SPHK1 knockdown protected against vitiligo via the regulation of FHL2.

Genome-wide identification and expression analysis of the BZR gene family in Zanthoxylum armatum DC and functional analysis of ZaBZR1 in drought tolerance.

MAIN CONCLUSION: In this study, six ZaBZRs were identified in Zanthoxylum armatum DC, and all the ZaBZRs were upregulated by abscisic acid (ABA) and drought. Overexpression of ZaBZR1 enhanced the drought tolerance of transgenic Nicotiana benthamian. Brassinosteroids (BRs) are a pivotal class of sterol hormones in plants that play a crucial role in plant growth and development. BZR (brassinazole resistant) is a crucial transcription factor in the signal transduction pathway of BRs. However, the BZR gene family members have not yet been identified in Zanthoxylum armatum DC. In this study, six members of the ZaBZR family were identified by bioinformatic methods. All six ZaBZRs exhibited multiple phosphorylation sites. Phylogenetic and collinearity analyses revealed a closest relationship between ZaBZRs and ZbBZRs located on the B subgenomes. Expression analysis revealed tissue-specific expression patterns of ZaBZRs in Z. armatum, and their promoter regions contained cis-acting elements associated with hormone response and stress induction. Additionally, all six ZaBZRs showed upregulation upon treatment after abscisic acid (ABA) and polyethylene glycol (PEG), indicating their participation in drought response. Subsequently, we conducted an extensive investigation of ZaBZR1. ZaBZR1 showed the highest expression in the root, followed by the stem and terminal bud. Subcellular localization analysis revealed that ZaBZR1 is present in the cytoplasm and nucleus. Overexpression of ZaBZR1 in transgenic Nicotiana benthamiana improved seed germination rate and root growth under drought conditions, reducing water loss rates compared to wild-type plants. Furthermore, ZaBZR1 increased proline content (PRO) and decreased malondialdehyde content (MDA), indicating improved tolerance to drought-induced oxidative stress. The transgenic plants also showed a reduced accumulation of reactive oxygen species. Importantly, ZaBZR1 up-regulated the expression of drought-related genes such as NbP5CS1, NbDREB2A, and NbWRKY44. These findings highlight the potential of ZaBZR1 as a candidate gene for enhancing drought resistance in transgenic N. benthamiana and provide insight into the function of ZaBZRs in Z. armatum.

OsNAC121 regulates root development, tillering, panicle morphology, and grain filling in rice plant.

Transcription factors in coordination with phytohormones form an intricate regulatory network modulating vital cellular mechanisms like development, growth and senescence in plants. In this study, we have functionally characterized the transcription factor OsNAC121 by developing gene silencing and overexpressing transgenic rice plants, followed by detailed analyses of the plant architecture. Transgenic lines exhibited remodelling in crown root development, lateral root structure and density, tiller height and number, panicle and grain morphologies, underpinning the imbalanced auxin: cytokinin ratio due to perturbed auxin transportation. Application of cytokinin, auxin and abscisic acid increased OsNAC121 gene expression nearly 17-, 6- and 91-folds, respectively. qRT-PCR results showed differential expressions of auxin and cytokinin pathway genes, implying their altered levels. A 47-fold higher expression level of OsNAC121 during milky stage in untransformed rice, compared to 14-day old shoot tissue, suggests its crucial role in grain filling; as evidenced by a large number of undeveloped grains produced by the gene silenced lines. Crippled gravitropic response by the transgenic plants indicates their impaired auxin transport. Bioinformatics revealed that OsNAC121 interacts with co-repressor (TOPLESS) proteins and forms a part of the inhibitor complex OsIAA10, an essential core component of auxin signalling pathway. Therefore, OsNAC121 emerges as an important regulator of various aspects of plant architecture through modulation of crosstalk between auxin and cytokinin, altering their concentration gradient in the meristematic zones, and consequently modifying different plant organogenesis processes.

Role of ZNF334 in cervical cancer: implications for EMT reversal and tumor suppression.

Zinc-finger proteins are involved in many biological processes. However, the role of Zinc-finger protein 334 (ZNF334) in cervical cancer remains unidentified. This study showed that promoter methylation of ZNF334 was responsible for its reduced expression. ZNF334 suppressed malignant biological behaviors in cervical cancer. Notably, ZNF334 reversed the EMT process both in vitro and in vivo. RNA-seq coupled with bioinformatics analysis caught P3H3 which is upregulated by ZNF334. Dual-luciferase reporter and Chromatin immunoprecipitation assays illustrated that ZNF334 directly regulate P3H3. Knockdown of P3H3 attenuated the reversal of EMT induced by ZNF334. Additionally, ZNF334 overexpression sensitized cervical cancer cells to the cytotoxic effects of paclitaxel, cyclosporine and sunitinib. In conclusions, this study illustrated that DNA methylation-based silencing ZNF334 played a vital role in cervical cancer, by regulating P3H3 in turn affects EMT. ZNF334 has the potential to become a novel diagnostic biomarker and a potential treatment target for cervical cancer.

YAP represses the TEAD-NF-κB complex and inhibits the growth of clear cell renal cell carcinoma.

The Hippo pathway is generally understood to inhibit tumor growth by phosphorylating the transcriptional cofactor YAP to sequester it to the cytoplasm and reduce the formation of YAP-TEAD transcriptional complexes. Aberrant activation of YAP occurs in various cancers. However, we found a tumor-suppressive function of YAP in clear cell renal cell carcinoma (ccRCC). Using cell cultures, xenografts, and patient-derived explant models, we found that the inhibition of upstream Hippo-pathway kinases MST1 and MST2 or expression of a constitutively active YAP mutant impeded ccRCC proliferation and decreased gene expression mediated by the transcription factor NF-κB. Mechanistically, the NF-κB subunit p65 bound to the transcriptional cofactor TEAD to facilitate NF-κB-target gene expression that promoted cell proliferation. However, by competing for TEAD, YAP disrupted its interaction with NF-κB and prompted the dissociation of p65 from target gene promoters, thereby inhibiting NF-κB transcriptional programs. This cross-talk between the Hippo and NF-κB pathways in ccRCC suggests that targeting the Hippo-YAP axis in an atypical manner-that is, by activating YAP-may be a strategy for slowing tumor growth in patients.

Pseudogenization of NK3 homeobox 2 (Nkx3.2) in monotremes provides insight into unique gastric anatomy and physiology.

The enzymatic breakdown and regulation of food passage through the vertebrate antral stomach and pyloric sphincter (antropyloric region) is a trait conserved over 450 million years. Development of the structures involved is underpinned by a highly conserved signalling pathway involving the hedgehog, bone morphogenetic protein and Wingless/Int-1 (Wnt) protein families. Monotremes are one of the few vertebrate lineages where acid-based digestion has been lost, and this is consistent with the lack of genes for hydrochloric acid secretion and gastric enzymes in the genomes of the platypus (Ornithorhynchus anatinus) and short-beaked echidna (Tachyglossus aculeatus) . Furthermore, these species feature unique gastric phenotypes, both with truncated and aglandular antral stomachs and the platypus with no pylorus. Here, we explore the genetic underpinning of monotreme gastric phenotypes, investigating genes important in antropyloric development using the newest monotreme genomes (mOrnAna1.pri.v4 and mTacAcu1) together with RNA-seq data. We found that the pathway constituents are generally conserved, but surprisingly, NK3 homeobox 2 (Nkx3.2) was pseudogenized in both platypus and echidna. We speculate that the unique sequence evolution of Grem1 and Bmp4 sequences in the echidna lineage may correlate with their pyloric-like restriction and that the convergent loss of gastric acid and stomach size genotypes and phenotypes in teleost and monotreme lineages may be a result of eco-evolutionary dynamics. These findings reflect the effects of gene loss on phenotypic evolution and further elucidate the genetic control of monotreme stomach anatomy and physiology.

Proton-pumping photoreceptor controls expression of ABC transporter by regulating transcription factor through light.

Light is a significant factor for living organisms with photosystems, like microbial rhodopsin-a retinal protein that functions as an ion pump, channel, and sensory transduction. Gloeobacter violaceus PCC7421, has a proton-pumping rhodopsin gene, the Gloeobacter rhodopsin (GR). The helix-turn-helix family of transcriptional regulators has various motifs, and they regulate gene expression in the presence of various metal ions. Here, we report that active proton outward pumping rhodopsin interacted with the helix-turn-helix transcription regulator and regulated gene expression. This interaction is confirmed using ITC analysis (KD of 8 µM) and determined the charged residues required. During in vitro experiments using fluorescent and luciferase reporter systems, ATP-binding cassette (ABC) transporters and the self-regulation of G. violaceus transcriptional regulator (GvTcR) are regulated by light, and gene regulation is observed in G. violaceus using the real-time polymerase chain reaction. These results expand our understanding of the natural potential and limitations of microbial rhodopsin function.

CaLAP1 and CaLAP2 orchestrate anthocyanin biosynthesis in the seed coat of Cicer arietinum.

MAIN CONCLUSION: Our findings shed light on the regulation of anthocyanin and proanthocyanidin biosynthesis in chickpea seed coats. Expression of R2R3-MYB transcription factors CaLAP1 and CaLAP2 enhanced the anthocyanins and proanthocyanidins content in chickpea. The seed coat color is a major economic trait in leguminous crop chickpea (Cicer arietinum). Anthocyanins and proanthocyanidins (PAs) are two classes of flavonoids that mainly contribute to the flower, seed coat and color of Desi chickpea cultivars. Throughout the land plant lineage, the accumulation of anthocyanins and PAs is regulated by MYB and bHLH transcription factors (TFs), which form an MBW (MYB, bHLH, and WD40) complex. Here, we report two R2R3-MYB TFs in chickpea belonging to the anthocyanin-specific subgroup-6, CaLAP1 (Legume Anthocyanin Production 1), and CaLAP2 (Legume Anthocyanin Production 2), which are mainly expressed in the flowers and developmental stages of the seeds. CaLAP1 and CaLAP2 interact with TT8-like CabHLH1 and WD40, forming the MBW complex, and bind to the promoter sequences of anthocyanin- and PA biosynthetic genes CaCHS6, CaDFR2, CaANS, and CaANR, leading to anthocyanins and PA accumulation in the seed coat of chickpea. Moreover, these CaLAPs partially complement the anthocyanin-deficient phenotype in the Arabidopsis thaliana sextuple mutant seedlings. Overexpression of CaLAPs in chickpea resulted in significantly higher expression of anthocyanin and PA biosynthetic genes leading to a darker seed coat color with higher accumulation of anthocyanin and PA. Our findings show that CaLAPs positively modulate anthocyanin and PA content in seed coats, which might influence plant development and resistance to various biotic and abiotic stresses.