Association of eating habits with mitochondrial DNA copy number in eveningness chronotypes: Origin research.

OBJECTIVE: To determine the relationship between eating habits and mitochondrial deoxyribonucleic acid copy number in adult cases of eveningness chronotypes. Methods: The cross-sectional, analytical study was conducted from September 2022 to June 2023 at the Physiology Department of the Islamic International Medical College, Rawalpindi, in collaboration with the Genetic Resource Centre, Rawalpindi, Pakistan, and comprised adult subjects who were assessed using the Morningness-Eveningness Questionnaire. The participants' eating habits were assessed using the Healthy Eating Assessment Questionnaire, and on they were divided into those with healthy eating habits in group A and those with unhealthy eating habits in group B. Deoxyribonucleic acid was extracted using the Chelex method, the mitochondrial deoxyribonucleic acid copy number of all participants was quantified using quantitative polymerase chain reaction. Data was analysed using SPSS 27. RESULTS: Of the 80 subjects, 30(37.5%) were males and 50(62.5%) were females. The overall mean age was 24.27±6.91 years (range: 18-45 years). There were 40(50%) subjects in each group. The mean mitochondrial deoxyribonucleic acid copy number in group A was 2.74±0.14 compared to 2.26±0.25 in group B (p<0.001). Conclusion: Subjects with healthy eating habits exhibited higher mitochondrial deoxyribonucleic acid copy numbers, indicating reduced damage to mitochondrial deoxyribonucleic acid.

Environmental circadian disruption re-writes liver circadian proteomes.

Circadian gene expression is fundamental to the establishment and functions of the circadian clock, a cell-autonomous and evolutionary-conserved timing system. Yet, how it is affected by environmental-circadian disruption (ECD) such as shiftwork and jetlag are ill-defined. Here, we provided a comprehensive and comparative description of male liver circadian gene expression, encompassing transcriptomes, whole-cell proteomes and nuclear proteomes, under normal and after ECD conditions. Under both conditions, post-translation, rather than transcription, is the dominant contributor to circadian functional outputs. After ECD, post-transcriptional and post-translational processes are the major contributors to whole-cell or nuclear circadian proteome, respectively. Furthermore, ECD re-writes the rhythmicity of 64% transcriptome, 98% whole-cell proteome and 95% nuclear proteome. The re-writing, which is associated with changes of circadian regulatory cis-elements, RNA-processing and protein localization, diminishes circadian regulation of fat and carbohydrate metabolism and persists after one week of ECD-recovery.

Clock gene homologs lin-42 and kin-20 regulate circadian rhythms in C. elegans.

Circadian rhythms are endogenous oscillations in nearly all organisms, from prokaryotes to humans, allowing them to adapt to cyclical environments for close to 24 h. Circadian rhythms are regulated by a central clock, based on a transcription-translation feedback loop. One important protein in the central loop in metazoan clocks is PERIOD, which is regulated in part by Casein kinase 1ε/δ (CK1ε/δ) phosphorylation. In the nematode Caenorhabditis elegans, period and casein kinase 1ε/δ are conserved as lin-42 and kin-20, respectively. Here, we studied the involvement of lin-42 and kin-20 in the circadian rhythms of the adult nematode using a bioluminescence-based circadian transcriptional reporter. We show that mutations of lin-42 and kin-20 generate a significantly longer endogenous period, suggesting a role for both genes in the nematode circadian clock, as in other organisms. These phenotypes can be partially rescued by overexpression of either gene under their native promoter. Both proteins are expressed in neurons and epidermal seam cells, as well as in other cells. Depletion of LIN-42 and KIN-20, specifically in neuronal cells after development, was sufficient to lengthen the period of oscillating sur-5 expression. Therefore, we conclude that LIN-42 and KIN-20 are critical regulators of the adult nematode circadian clock through neuronal cells.

Circadian rhythm disruption-mediated downregulation of Bmal1 exacerbates DSS-induced colitis by impairing intestinal barrier.

Background: Circadian rhythm disruption (CRD) is thought to increase the risk of inflammatory bowel disease. The deletion of Bmal1, a core transcription factor, leads to a complete loss of the circadian rhythm and exacerbates the severity of dextran sodium sulfate (DSS)-induced colitis in mice. However, the underlying mechanisms by which CRD and Bmal1 mediate IBD are still unclear. Methods: We used a CRD mouse model, a mouse colitis model, and an in vitro model of colonic epithelial cell monolayers. We also knocked down and overexpressed Bmal1 in Caco-2 cells by transfecting lentivirus in vitro. The collected colon tissue and treated cells were assessed and analyzed using immunohistochemistry, immunofluorescence staining, quantitative reverse transcription-polymerase chain reaction, western blot, flow cytometry, transmission electron microscopy, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling staining. Results: We found that CRD mice with downregulated Bmal1 expression were more sensitive to DSS-induced colitis and had more severely impaired intestinal barrier function than wild-type mice. Bmal1-/- mice exhibited more severe colitis, accompanied by decreased tight junction protein levels and increased apoptosis of intestinal epithelial cells compared with wild-type mice, which were alleviated by using the autophagy agonist rapamycin. Bmal1 overexpression attenuated Lipopolysaccharide-induced apoptosis of intestinal epithelial cells and impaired intestinal epithelial cells barrier function in vitro, while inhibition of autophagy reversed this protective effect. Conclusion: This study suggests that CRD leads to the downregulation of Bmal1 expression in the colon, which may exacerbate DSS-induced colitis in mice, and that Bmal1 may serve as a novel target for treating inflammatory bowel disease.

Circadian rhythm and host genetics.

This chapter aims to explore the usefulness of the latest advances in genetic studies in the field of the circadian system in the future development of individualised strategies for health improvement based on lifestyle intervention. Due to the multifactorial and complex nature of the circadian system, we focus on the highly prevalent phenotypes in the population that are key to understanding its biology from an evolutionary perspective and that can be modulated by lifestyle. Therefore, we leave in the background those phenotypes that constitute infrequent pathologies or in which the current level of scientific evidence does not favour the implementation of practical approaches of this type. Therefore, from an evolutionary paradigm, this chapter addresses phenotypes such as morning chronotypes, evening chronotypes, extreme chronotypes, and other key concepts such as circadian rhythm amplitude, resilience to changes in circadian rhythm, and their relationships with pathologies associated with circadian rhythm imbalances.

Sleep regulation and host genetics.

Due to the multifactorial and complex nature of rest, we focus on phenotypes related to sleep. Sleep regulation is a multifactorial process. In this chapter, we focus on those phenotypes inherent to sleep that are highly prevalent in the population, and that can be modulated by lifestyle, such as sleep quality and duration, insomnia, restless leg syndrome and daytime sleepiness. We, therefore, leave in the background those phenotypes that constitute infrequent pathologies or for which the current level of scientific evidence does not favour the implementation of practical approaches of this type. Similarly, the regulation of sleep quality is intimately linked to the regulation of the circadian rhythm. Although this relationship is discussed in the sections that require it, the in-depth study of circadian rhythm regulation at the molecular level deserves a separate chapter, and this is how it is dealt with in this volume.

Circadian-driven tissue specificity is constrained under caloric restricted feeding conditions.

Tissue specificity is a fundamental property of an organ that affects numerous biological processes, including aging and longevity, and is regulated by the circadian clock. However, the distinction between circadian-affected tissue specificity and other tissue specificities remains poorly understood. Here, using multi-omics data on circadian rhythms in mice, we discovered that approximately 35% of tissue-specific genes are directly affected by circadian regulation. These circadian-affected tissue-specific genes have higher expression levels and are associated with metabolism in hepatocytes. They also exhibit specific features in long-reads sequencing data. Notably, these genes are associated with aging and longevity at both the gene level and at the network module level. The expression of these genes oscillates in response to caloric restricted feeding regimens, which have been demonstrated to promote longevity. In addition, aging and longevity genes are disrupted in various circadian disorders. Our study indicates that the modulation of circadian-affected tissue specificity is essential for understanding the circadian mechanisms that regulate aging and longevity at the genomic level.

Circadian Rhythm Disruption in Hepatocellular Carcinoma Investigated by Integrated Analysis of Bulk and Single-Cell RNA Sequencing Data.

Circadian rhythms are essential regulators of a multitude of physiological and behavioral processes, such as the metabolism and function of the liver. Circadian rhythms are crucial to liver homeostasis, as the liver is a key metabolic organ accountable for the systemic equilibrium of the body. Circadian rhythm disruption alone is sufficient to cause liver cancer through the maintenance of hepatic metabolic disorder. Although there is evidence linking CRD to hepatocarcinogenesis, the precise cellular and molecular mechanisms that underlie the circadian crosstalk that leads to hepatocellular carcinoma remain unknown. The expression of CRD-related genes in HCC was investigated in this study via bulk RNA transcriptomic analysis and single-cell sequencing. Dysregulated CRD-related genes are predominantly found in hepatocytes and fibroblasts, according to the findings. By using a combination of single-cell RNA sequencing and bulk RNA sequencing analyses, the dysregulated CRD-related genes ADAMTS13, BIRC5, IGFBP3, MARCO, MT2A, NNMT, and PGLYRP2 were identified. The survival analysis using the Kaplan-Meier method revealed a significant correlation between the expression levels of BIRC5 and IGFBP3 and the survival of patients diagnosed with HCC.

Revealing the profound influence of diapause on gene expression: Insights from the annual transcriptome of the copepod Calanus finmarchicus.

Annual rhythms are observed in living organisms with numerous ecological implications. In the zooplanktonic copepod Calanus finmarchicus, such rhythms are crucial regarding its phenology, body lipid accumulation, and global carbon storage. Climate change drives annual biological rhythms out of phase with the prevailing environmental conditions with yet unknown but potentially catastrophic consequences. However, the molecular dynamics underlying phenology are still poorly described. In a rhythmic analysis of C. finmarchicus annual gene expression, results reveal that more than 90% of the transcriptome shows significant annual rhythms, with abrupt and dramatic upheaval between the active and diapause life cycle states. This work explores the implication of the circadian clock in the annual timing, which may control epigenetic mechanisms to profoundly modulate gene expression in response to calendar time. Results also suggest an increased light sensitivity during diapause that would ensure the photoperiodic entrainment of the endogenous annual clock.

Circadian patterns of growth factor receptor-dependent signaling and implications for carcinogenesis.

Several different signaling pathways that regulate cell proliferation and differentiation are initiated by binding of ligands to cell-surface and membrane-bound enzyme-linked receptors, such as receptor tyrosine kinases and serine-threonine kinases. They prompt phosphorylation of tyrosine and serine-threonine residues and initiate downstream signaling pathways and priming of intracellular molecules that convey the signal in the cytoplasm and nucleus, with transcriptional activation of specific genes enriching cell growth and survival-related cascades. These cell processes are rhythmically driven by molecular clockworks endowed in every cell type and when deregulated play a crucial role in cancer onset and progression. Growth factors and their matching receptor-dependent signaling are frequently overexpressed and/or dysregulated in many cancer types. In this review we focus on the interplay between biological clocks and Growth Factor Receptor-dependent signaling in the context of carcinogenesis.

Metabolic Profile and Lipid Metabolism Phenotype in Mice with Conditional Deletion of Hepatic BMAL1.

The disruption of circadian rhythms (CRs) has been linked to metabolic disorders, yet the role of hepatic BMAL1, a key circadian regulator, in the whole-body metabolism and the associated lipid metabolic phenotype in the liver remains unclear. Bmal1 floxed (Bmal1f/f) and hepatocyte-specific Bmal1 knockout (Bmal1hep-/-) C57BL/6J mice underwent a regular feeding regimen. Hepatic CR, lipid content, mitochondrial function, and systemic metabolism were assessed at zeitgeber time (ZT) 0 and ZT12. Relevant molecules were examined to elucidate the metabolic phenotype. Hepatocyte-specific knockout of Bmal1 disrupted the expression of rhythmic genes in the liver. Bmal1hep-/- mice exhibited decreased hepatic TG content at ZT0, primarily due to enhanced lipolysis, reduced lipogenesis, and diminished lipid uptake. The ß-oxidation function of liver mitochondria decreased at both ZT0 and ZT12. Our findings on the metabolic profile and associated hepatic lipid metabolism in the absence of Bmal1 in hepatocytes provides new insights into metabolic syndromes from the perspective of liver CR disturbances.

Clock-dependent chromatin accessibility rhythms regulate circadian transcription.

Chromatin organization plays a crucial role in gene regulation by controlling the accessibility of DNA to transcription machinery. While significant progress has been made in understanding the regulatory role of clock proteins in circadian rhythms, how chromatin organization affects circadian rhythms remains poorly understood. Here, we employed ATAC-seq (Assay for Transposase-Accessible Chromatin with Sequencing) on FAC-sorted Drosophila clock neurons to assess genome-wide chromatin accessibility at dawn and dusk over the circadian cycle. We observed significant oscillations in chromatin accessibility at promoter and enhancer regions of hundreds of genes, with enhanced accessibility either at dusk or dawn, which correlated with their peak transcriptional activity. Notably, genes with enhanced accessibility at dusk were enriched with E-box motifs, while those more accessible at dawn were enriched with VRI/PDP1-box motifs, indicating that they are regulated by the core circadian feedback loops, PER/CLK and VRI/PDP1, respectively. Further, we observed a complete loss of chromatin accessibility rhythms in per01 null mutants, with chromatin consistently accessible at both dawn and dusk, underscoring the critical role of Period protein in driving chromatin compaction during the repression phase at dawn. Together, this study demonstrates the significant role of chromatin organization in circadian regulation, revealing how the interplay between clock proteins and chromatin structure orchestrates the precise timing of biological processes throughout the day. This work further implies that variations in chromatin accessibility might play a central role in the generation of diverse circadian gene expression patterns in clock neurons.

Mice with deficiency in Pcdh15, a gene associated with bipolar disorders, exhibit significantly elevated diurnal amplitudes of locomotion and body temperature.

Genetic factors significantly affect the pathogenesis of psychiatric disorders. However, the specific pathogenic mechanisms underlying these effects are not fully understood. Recent extensive genomic studies have implicated the protocadherin-related 15 (PCDH15) gene in the onset of psychiatric disorders, such as bipolar disorder (BD). To further investigate the pathogenesis of these psychiatric disorders, we developed a mouse model lacking Pcdh15. Notably, although PCDH15 is primarily identified as the causative gene of Usher syndrome, which presents with visual and auditory impairments, our mice with Pcdh15 homozygous deletion (Pcdh15-null) did not exhibit observable structural abnormalities in either the retina or the inner ear. The Pcdh15-null mice showed very high levels of spontaneous motor activity which was too disturbed to perform standard behavioral testing. However, the Pcdh15 heterozygous deletion mice (Pcdh15-het) exhibited enhanced spontaneous locomotor activity, reduced prepulse inhibition, and diminished cliff avoidance behavior. These observations agreed with the symptoms observed in patients with various psychiatric disorders and several mouse models of psychiatric diseases. Specifically, the hyperactivity may mirror the manic episodes in BD. To obtain a more physiological, long-term quantification of the hyperactive phenotype, we implanted nano tag® sensor chips in the animals, to enable the continuous monitoring of both activity and body temperature. During the light-off period, Pcdh15-null exhibited elevated activity and body temperature compared with wild-type (WT) mice. However, we observed a decreased body temperature during the light-on period. Comprehensive brain activity was visualized using c-Fos mapping, which was assessed during the activity and temperature peak and trough. There was a stark contrast between the distribution of c-Fos expression in Pcdh15-null and WT brains during both the light-on and light-off periods. These results provide valuable insights into the neural basis of the behavioral and thermal characteristics of Pcdh15-deletion mice. Therefore, Pcdh15-deletion mice can be a novel model for BD with mania and other psychiatric disorders, with a strong genetic component that satisfies both construct and surface validity.

Sex-specific associations between circadian-related genes and depression in UK Biobank participants highlight links to glucose metabolism, inflammation and neuroplasticity pathways.

Depressive disorders have increased in global prevalence, making improved management of these disorders a public health priority. Prior research has linked circadian clock genes to depression, either through direct interactions with mood-related pathways in the brain or by modulating the phase of circadian rhythms. Using machine learning and statistical techniques, we explored associations between 157,347 SNP variants from 51 circadian-related genes and depression scores from the patient health questionnaire 9 (PHQ-9) in 99,939 UK Biobank participants. Our results highlight multiple pathways linking the circadian system to mood, including metabolic, monoamine, immune, and stress-related pathways. Notably, genes regulating glucose metabolism and inflammation (GSK3B, LEP, RORA, and NOCT) were prominent factors in females, in addition to DELEC1 and USP46, two genes of unknown function. In contrast, FBXL3 and DRD4 emerged as significant risk factors for male depression. We also found epistatic interactions involving RORA, NFIL3, and ZBTB20 as either risk or protective factors for depression, underscoring the importance of transcription factors (ZBTB20, NFIL3) and hormone receptors (RORA) in depression etiology. Understanding the complex, sex-specific links between circadian genes and mood disorders will facilitate the development of therapeutic interventions and enhance the efficacy of multi-target treatments for depression.

Circadian transcriptome of pancreatic adenocarcinoma unravels chronotherapeutic targets.

Pancreatic ductal adenocarcinoma (PDA) is a lethal cancer characterized by a poor outcome and an increasing incidence. A significant majority (>80%) of newly diagnosed cases are deemed unresectable, leaving chemotherapy as the sole viable option, though with only moderate success. This necessitates the identification of improved therapeutic options for PDA. We hypothesized that there are temporal variations in cancer-relevant processes within PDA tumors, offering insights into the optimal timing of drug administration - a concept termed chronotherapy. In this study, we explored the presence of the circadian transcriptome in PDA using patient-derived organoids and validated these findings by comparing PDA data from The Cancer Genome Atlas with noncancerous healthy pancreas data from GTEx. Several PDA-associated pathways (cell cycle, stress response, Rho GTPase signaling) and cancer driver hub genes (EGFR and JUN) exhibited a cancer-specific rhythmic pattern intricately linked to the circadian clock. Through the integration of multiple functional measurements for rhythmic cancer driver genes, we identified top chronotherapy targets and validated key findings in molecularly divergent pancreatic cancer cell lines. Testing the chemotherapeutic efficacy of clinically relevant drugs further revealed temporal variations that correlated with drug-target cycling. Collectively, our study unravels the PDA circadian transcriptome and highlights a potential approach for optimizing chrono-chemotherapeutic efficacy.

Circadian and photoperiodic regulation of the vegetative to reproductive transition in plants.

As sessile organisms, plants must respond constantly to ever-changing environments to complete their life cycle; this includes the transition from vegetative growth to reproductive development. This process is mediated by photoperiodic response to sensing the length of night or day through circadian regulation of light-signaling molecules, such as phytochromes, to measure the length of night to initiate flowering. Flowering time is the most important trait to optimize crop performance in adaptive regions. In this review, we focus on interplays between circadian and light signaling pathways that allow plants to optimize timing for flowering and seed production in Arabidopsis, rice, soybean, and cotton. Many crops are polyploids and domesticated under natural selection and breeding. In response to adaptation and polyploidization, circadian and flowering pathway genes are epigenetically reprogrammed. Understanding the genetic and epigenetic bases for photoperiodic flowering will help improve crop yield and resilience in response to climate change.

tauFisher predicts circadian time from a single sample of bulk and single-cell pseudobulk transcriptomic data.

As the circadian clock regulates fundamental biological processes, disrupted clocks are often observed in patients and diseased tissues. Determining the circadian time of the patient or the tissue of focus is essential in circadian medicine and research. Here we present tauFisher, a computational pipeline that accurately predicts circadian time from a single transcriptomic sample by finding correlations between rhythmic genes within the sample. We demonstrate tauFisher's performance in adding timestamps to both bulk and single-cell transcriptomic samples collected from multiple tissue types and experimental settings. Application of tauFisher at a cell-type level in a single-cell RNAseq dataset collected from mouse dermal skin implies that greater circadian phase heterogeneity may explain the dampened rhythm of collective core clock gene expression in dermal immune cells compared to dermal fibroblasts. Given its robustness and generalizability across assay platforms, experimental setups, and tissue types, as well as its potential application in single-cell RNAseq data analysis, tauFisher is a promising tool that facilitates circadian medicine and research.

CLOCK evolved in cnidaria to synchronize internal rhythms with diel environmental cues.

The circadian clock enables anticipation of the day/night cycle in animals ranging from cnidarians to mammals. Circadian rhythms are generated through a transcription-translation feedback loop (TTFL or pacemaker) with CLOCK as a conserved positive factor in animals. However, CLOCK's functional evolutionary origin and mechanism of action in basal animals are unknown. In the cnidarian Nematostella vectensis, pacemaker gene transcript levels, including NvClk (the Clock ortholog), appear arrhythmic under constant darkness, questioning the role of NvCLK. Utilizing CRISPR/Cas9, we generated a NvClk allele mutant (NvClkΔ), revealing circadian behavior loss under constant dark (DD) or light (LL), while maintaining a 24 hr rhythm under light-dark condition (LD). Transcriptomics analysis revealed distinct rhythmic genes in wild-type (WT) polypsunder LD compared to DD conditions. In LD, NvClkΔ/Δ polyps exhibited comparable numbers of rhythmic genes, but were reduced in DD. Furthermore, under LD, the NvClkΔ/Δ polyps showed alterations in temporal pacemaker gene expression, impacting their potential interactions. Additionally, differential expression of non-rhythmic genes associated with cell division and neuronal differentiation was observed. These findings revealed that a light-responsive pathway can partially compensate for circadian clock disruption, and that the Clock gene has evolved in cnidarians to synchronize rhythmic physiology and behavior with the diel rhythm of the earth's biosphere.

Transcriptomic analysis of AHR wildtype and Knock-out rat livers supports TCDD's role in AHR/ARNT-mediated circadian disruption and hepatotoxicity.

Single, high doses of TCDD in rats are known to cause wasting, a progressive loss of 30 to 50% body weight and death within several weeks. To identify pathway perturbations at or near doses causing wasting, we examined differentially gene expression (DGE) and pathway enrichment in centrilobular (CL) and periportal (PP) regions of female rat livers following 6 dose levels of TCDD - 0, 3, 22, 100, 300, and 1000 ng/kg/day, 5 days/week for 4 weeks. At the higher doses, rats lost weight, had increased liver/body weight ratios and nearly complete cessation of liver cell proliferation, signs consistent with wasting. DGE curves were left shifted for the CL versus the PP regions. Canonical Phase I and Phase II genes were maximally increased at lower doses and remained elevated at all doses. At lower doses, ≤ 22 ng/kg/day in the CL and ≤ 100 ng/kg/day, upregulated genes showed transcription factor (TF) enrichment for AHR and ARNT. At the mid- and high-dose doses, there was a large number of downregulated genes and pathway enrichment for DEGs which showed downregulation of many cellular metabolism processes including those for steroids, fatty acid metabolism, pyruvate metabolism and citric acid cycle. There was significant TF enrichment of the hi-dose downregulated genes for RXR, ESR1, LXR, PPARalpha. At the highest dose, there was also pathway enrichment with upregulated genes for extracellular matrix organization, collagen formation, hemostasis and innate immune system. TCDD demonstrates most of its effects through binding the aryl hydrocarbon receptor (AHR) while the downregulation of metabolism genes at higher TCDD doses is known to be independent of AHR binding to DREs. Based on our results with DEG, we provide a hypothesis for wasting in which high doses of TCDD shift circadian processes away from the resting state, leading to greatly reduced synthesis of steroids and complex lipids needed for cell growth, and producing gene expression signals consistent with an epithelial-to-mesenchymal transition in hepatocytes.

Functional and regulatory diversity of homeobox-leucine zipper transcription factors BnaHB6 under dehydration and salt stress in Brassica napus L.

The plant-specific homeodomain-leucine zipper I subfamily is involved in the regulation of various biological processes, particularly growth, development and stress response. In the present study, we characterized four BnaHB6 homologues from Brassica napus. All BnaHB6 proteins have transcriptional activation activity. Structural and functional data indicate the complex role of BnaHB6 genes in regulating biological processes, with some functions conserved and others diverged. Transcriptional analyzes revealed that they are induced in a similar manner in different tissues but show different expression patterns in response to stress and circadian rhythm. Only the BnaA09HB6 and BnaC08HB6 genes are expressed under dehydration and salt stress, and in darkness. The partial transcriptional overlap of BnaHB6s with the evolutionarily related genes BnaHB5 and BnaHB16 was also observed. Transgenic Arabidopsis thaliana plants expressing a single proBnaHB6::GUS partially confirmed the expression results. Bioinformatic analysis allowed the identification of TF-binding sites in the BnaHB6 promoters that may control their expression under stress and circadian rhythm. ChIP-qPCR analysis revealed that BnaA09HB6 and BnaC08HB6 bind directly to the promoters of the target genes BnaABF4 and BnaDREB2A. Comparison of their expression patterns in the WT plants and the bnac08hb6 mutant showed that BnaC08HB6 positively regulates the expression of the BnaABF4 and BnaDREB2A genes under dehydration and salt stress. We conclude that four BnaHB6 homologues have distinct functions in response to stress despite high sequence similarity, possibly indicating different binding preferences with BnaABF4 and BnaDREB2A. We hypothesize that BnaC08HB6 and BnaA09HB6 function in a complex regulatory network under stress.