Drought response revealed by chromatin organization variation and transcriptional regulation in cotton.

BACKGROUND: Cotton is a major world cash crop and an important source of natural fiber, oil, and protein. Drought stress is becoming a restrictive factor affecting cotton production. To facilitate the development of drought-tolerant cotton varieties, it is necessary to study the molecular mechanism of drought stress response by exploring key drought-resistant genes and related regulatory factors. RESULTS: In this study, two cotton varieties, ZY007 (drought-sensitive) and ZY168 (drought-tolerant), showing obvious phenotypic differences under drought stress, were selected. A total of 25,898 drought-induced genes were identified, exhibiting significant enrichment in pathways related to plant stress responses. Under drought induction, At subgenome expression bias was observed at the whole-genome level, which may be due to stronger inhibition of Dt subgenome expression. A gene co-expression module that was significantly associated with drought resistance was identified. About 90% of topologically associating domain (TAD) boundaries were stable, and 6613 TAD variation events were identified between the two varieties under drought. We identified 92 genes in ZY007 and 98 in ZY168 related to chromatin 3D structural variation and induced by drought stress. These genes are closely linked to the cotton response to drought stress through canonical hormone-responsive pathways, modulation of kinase and phosphatase activities, facilitation of calcium ion transport, and other related molecular mechanisms. CONCLUSIONS: These results lay a foundation for elucidating the molecular mechanism of the cotton drought response and provide important regulatory locus and gene resources for the future molecular breeding of drought-resistant cotton varieties.

Precise fine-turning of GhTFL1 by base editing tools defines ideal cotton plant architecture.

BACKGROUND: CRISPR/Cas-derived base editor enables precise editing of target sites and has been widely used for basic research and crop genetic improvement. However, the editing efficiency of base editors at different targets varies greatly. RESULTS: Here, we develop a set of highly efficient base editors in cotton plants. GhABE8e, which is fused to conventional nCas9, exhibits 99.9% editing efficiency, compared to GhABE7.10 with 64.9%, and no off-target editing is detected. We further replace nCas9 with dCpf1, which recognizes TTTV PAM sequences, to broaden the range of the target site. To explore the functional divergence of TERMINAL FLOWER 1 (TFL1), we edit the non-coding and coding regions of GhTFL1 with 26 targets to generate a comprehensive allelic population including 300 independent lines in cotton. This allows hidden pleiotropic roles for GhTFL1 to be revealed and allows us to rapidly achieve directed domestication of cotton and create ideotype germplasm with moderate height, shortened fruiting branches, compact plant, and early-flowering. Further, by exploring the molecular mechanism of the GhTFL1L86P and GhTFL1K53G+S78G mutations, we find that the GhTFL1L86P mutation weakens the binding strength of the GhTFL1 to other proteins but does not lead to a complete loss of GhTFL1 function. CONCLUSIONS: This strategy provides an important technical platform and genetic information for the study and creation of ideal plant architecture.

GhL1L1 regulates the contents of unsaturated fatty acids by activating the expression of GhFAD2 genes in cotton.

Edible oils with high unsaturated fatty acids, particularly oleic acid, are beneficial to human health. Cotton is one of the top five oil crops in the world, but the mechanism of high-quality oil synthesis and regulatory networks in cotton are largely unclear. Here, we identified Leafy cotyledon1-like 1 (GhL1L1), a NF-YB subfamily gene that is specifically expressed during somatic embryogenesis and seed maturation in cotton. Overexpression of GhL1L1 regulates the contents of unsaturated fatty acids in cotton, especially in the seeds, which is associated with altered expression of the cotton fatty acid biosynthesis-related genes. GhL1L1 synergistically enhanced the expression of GhFAD2-1A by binding to the G-box in its promoter, leading to an increase in the content of linoleic acid. Furthermore, this activation could be enhanced by GhNF-YC2 and GhNF-YA1 by form a transcriptional complex. Collectively, these results contribute to provide new insights into the molecular mechanism of oil biosynthesis in cotton and can facilitate genetic manipulation of cotton varieties with enhanced oil content.

Geochemical Analysis of Dolomite in the Fourth Member of the Upper Sinian Dengying Formation, Northern Sichuan Basin, China.

The fourth member of the Sinian Dengying Formation in northern Sichuan was reformed by multistage diagenetic fluids. It is beneficial to systematically analyze the diagenetic evolution of the area to clarify the sedimentary evolution of the dolomite in the fourth member of the Dengying Formation and the fluid characteristics at different diagenetic stages. In this study, the petrological characteristics, vertical sedimentary evolution, diagenetic fluid stages, and diagenetic environment of Dengsi dolomite were analyzed by using carbon-oxygen strontium isotopes, rare-earth elements, major and trace elements, combined with the supporting thin section identification and cathodoluminescence techniques, and the source and properties of diagenetic fluid of Dengsi dolomite in different diagenetic environments were determined. The results show that (1) during the deposition period of the fourth member of the Dengying Formation, the water body showed a trend from shallow to deep and then steadily to shallow, which was in a shallow freshwater reducing environment and had a warm and humid paleoclimate environment; (2) the dolomite of the fourth member of Dengying Formation in northern Sichuan is less affected by terrigenous materials. The C and O isotopes of dolomite are generally negative at the top of the fourth member of the Dengying Formation, and the top of the formation is obviously transformed into rock by atmospheric fresh water; rare-earth elements show that dolomitized fluid is dominated by primitive seawater, and the marine fluid from or flowing through the carbonaceous shale formation rich in feldspar minerals in the Qiongzhusi formation during burial period makes the rare earth elements show obvious positive EU anomalies. The high 87Sr/86Sr ratio of the surrounding rock and cement is caused by the 87Sr-rich marine fluid coming from the fault communication or along the unconformity zone; (3) since the deposition of the dolomite of the fourth member of Dengshan formation in northern Sichuan, it has experienced multistage diagenesis. The multistage cement and fillings in the pores have recorded at least seven stages of fluid activity history: during the quasi-contemporaneous period, fresh water was transformed to increase pores, and seawater was cemented to damage pores. During the supergene period, the pore size of atmospheric freshwater increased generally; during the middle deep burial period, the acid fluid was reformed to increase the porosity, the 87Sr-rich hydrocarbon-bearing fluid was used to fill the damaged pores, and the Eu2+-rich marine source fluid was used to fill the damaged pores. In the deep burial period, siliceous hydrothermal fluid filled the damaged hole.

Genome-wide identification and expression analysis of PYL family genes and functional characterization of GhPYL8D2 under drought stress in Gossypium hirsutum.

Cotton is a crucial economic crop, serving as a natural fiber source for the textile industry. However, drought stress poses a significant threat to cotton fiber quality and productivity worldwide. Pyrabactin Resistance 1-Like (PYL) proteins, as abscisic acid (ABA) receptors, play a crucial role in adverse stress responses, but knowledge about the PYLs in cotton remains limited. In our study, we identified 40 GhPYL genes in Gossypium hirsutum through a genome-wide analysis of the cotton genome database. Our analysis revealed that the PYL family formed three distinct subfamilies with typical family characteristics in G. hirsutum. Additionally, through quantitative expression analysis, including transcriptome dataset and qRT-PCR, we found that all GhPYLs were expressed in all tissues of G. hirsutum, and all GhPYLs were differentially expressed under drought stress. Among them, GhPYL4A1, GhPY5D1, GhPY8D2, and a member of the type 2C protein phosphatases clade A family in Gossypium hirsutum (GhPP2CA), GhHAI2D, showed significant differences in expression levels within 12 h after stress treatment. Our protein interaction analysis and BiFC demonstrated the complex regulatory network between GhPYL family proteins and GhPP2CA proteins. We also found that there is an interaction between GhPYL8D2 and GhHAI2D, and through drought treatment of transgenic cotton, we found that GhPYL8D2 played a vital role in the response of G. hirsutum to drought through stomatal control via co-regulation with GhHAI2D. Our findings provide useful insights into the regulation of GhPYL family genes that occur in response to abiotic stresses in cotton.

Stochastic modeling of the mRNA life process: A generalized master equation.

The mRNA life cycle is a complex biochemical process, involving transcription initiation, elongation, termination, splicing, and degradation. Each of these molecular events is multistep and can create a memory. The effect of this molecular memory on gene expression is not clear, although there are many related yet scattered experimental reports. To address this important issue, we develop a general theoretical framework formulated as a master equation in the sense of queue theory, which can reduce to multiple previously studied gene models in limiting cases. This framework allows us to interpret experimental observations, extract kinetic parameters from experimental data, and identify how the mRNA kinetics vary under regulatory influences. Notably, it allows us to evaluate the influences of elongation processes on mature RNA distribution; e.g., we find that the non-exponential elongation time can induce the bimodal mRNA expression and there is an optimal elongation noise intensity such that the mature RNA noise achieves the lowest level. In a word, our framework can not only provide insight into complex mRNA life processes but also bridge a dialogue between theoretical studies and experimental data.

Comprehensive non-coding RNA analysis reveals specific lncRNA/circRNA-miRNA-mRNA regulatory networks in the cotton response to drought stress.

Root and leaf are essential organs of plants in sensing and responding to drought stress. However, comparative knowledge of non-coding RNAs (ncRNAs) of root and leaf tissues in the regulation of drought response in cotton is limited. Here, we used deep sequencing data of leaf and root tissues of drought-resistant and drought-sensitive cotton varieties for identifying miRNAs, lncRNAs and circRNAs. A total of 1531 differentially expressed (DE) ncRNAs was identified, including 77 DE miRNAs, 1393 DE lncRNAs and 61 DE circRNAs. The tissue-specific and variety-specific competing endogenous RNA (ceRNA) networks of DE lncRNA-miRNA-mRNA response to drought were constructed. Furthermore, the novel drought-responsive lncRNA 1 (DRL1), specifically and differentially expressed in root, was verified to positively affect phenotypes of cotton seedlings under drought stress, competitively binding to miR477b with GhNAC1 and GhSCL3. In addition, we also constructed another ceRNA network consisting of 18 DE circRNAs, 26 DE miRNAs and 368 DE mRNAs. Fourteen circRNA were characterized, and a novel molecular regulatory system of circ125- miR7484b/miR7450b was proposed under drought stress. Our findings revealed the specificity of ncRNA expression in tissue- and variety-specific patterns involved in the response to drought stress, and uncovered novel regulatory pathways and potentially effective molecules in genetic improvement for crop drought resistance.

Single-cell resolution analysis reveals the preparation for reprogramming the fate of stem cell niche in cotton lateral meristem.

BACKGROUND: Somatic embryogenesis is a major process for plant regeneration. However, cell communication and the gene regulatory network responsible for cell reprogramming during somatic embryogenesis are still largely unclear. Recent advances in single-cell technologies enable us to explore the mechanism of plant regeneration at single-cell resolution. RESULTS: We generate a high-resolution single-cell transcriptomic landscape of hypocotyl tissue from the highly regenerable cotton genotype Jin668 and the recalcitrant TM-1. We identify nine putative cell clusters and 23 cluster-specific marker genes for both cultivars. We find that the primary vascular cell is the major cell type that undergoes cell fate transition in response to external stimulation. Further developmental trajectory and gene regulatory network analysis of these cell clusters reveals that a total of 41 hormone response-related genes, including LAX2, LAX1, and LOX3, exhibit different expression patterns in the primary xylem and cambium region of Jin668 and TM-1. We also identify novel genes, including CSEF, PIS1, AFB2, ATHB2, PLC2, and PLT3, that are involved in regeneration. We demonstrate that LAX2, LAX1 and LOX3 play important roles in callus proliferation and plant regeneration by CRISPR/Cas9 editing and overexpression assay. CONCLUSIONS: This study provides novel insights on the role of the regulatory network in cell fate transition and reprogramming during plant regeneration driven by somatic embryogenesis.

Inferring transcriptional bursting kinetics from single-cell snapshot data using a generalized telegraph model.

Gene expression has inherent stochasticity resulting from transcription's burst manners. Single-cell snapshot data can be exploited to rigorously infer transcriptional burst kinetics, using mathematical models as blueprints. The classical telegraph model (CTM) has been widely used to explain transcriptional bursting with Markovian assumptions. However, growing evidence suggests that the gene-state dwell times are generally non-exponential, as gene-state switching is a multi-step process in organisms. Therefore, interpretable non-Markovian mathematical models and efficient statistical inference methods are urgently required in investigating transcriptional burst kinetics. We develop an interpretable and tractable model, the generalized telegraph model (GTM), to characterize transcriptional bursting that allows arbitrary dwell-time distributions, rather than exponential distributions, to be incorporated into the ON and OFF switching process. Based on the GTM, we propose an inference method for transcriptional bursting kinetics using an approximate Bayesian computation framework. This method demonstrates an efficient and scalable estimation of burst frequency and burst size on synthetic data. Further, the application of inference to genome-wide data from mouse embryonic fibroblasts reveals that GTM would estimate lower burst frequency and higher burst size than those estimated by CTM. In conclusion, the GTM and the corresponding inference method are effective tools to infer dynamic transcriptional bursting from static single-cell snapshot data.

N6-methyladenosine RNA modification regulates cotton drought response in a Ca2+ and ABA-dependent manner.

N6 -methyladenosine (m6 A) is the most prevalent internal modification present in mRNAs, and is considered to participate in a range of developmental and biological processes. Drought response is highly regulated at the genomic, transcriptional and post-transcriptional levels. However, the biological function and regulatory mechanism of m6 A modification in the drought stress response is still poorly understood. We generated a transcriptome-wide m6 A map using drought-resistant and drought-sensitive varieties of cotton under different water deficient conditions to uncover patterns of m6 A methylation in cotton response to drought stress. The results reveal that m6 A represents a common modification and exhibit dramatic changes in distribution during drought stress. More 5'UTR m6 A was deposited in the drought-resistant variety and was associated with a positive effect on drought resistance by regulating mRNA abundance. Interestingly, we observed that increased m6 A abundance was associated with increased mRNA abundance under drought, contributing to drought resistance, and vice versa. The demethylase GhALKBH10B was found to decrease m6 A levels, facilitating the mRNA decay of ABA signal-related genes (GhZEP, GhNCED4 and GhPP2CA) and Ca2+ signal-related genes (GhECA1, GhCNGC4, GhANN1 and GhCML13), and mutation of GhALKBH10B enhanced drought resistance at seedling stage in cotton. Virus-induced gene silencing (VIGS) of two Ca2+ -related genes, GhECA1 and GhCNGC4, reduced drought resistance with the decreased m6 A enrichment on silenced genes in cotton. Collectively, we reveal a novel mechanism of post-transcriptional modification involved in affecting drought response in cotton, by mediating m6 A methylation on targeted transcripts in the ABA and Ca2+ signalling transduction pathways.

MERISTEM-DEFECTIVE regulates the balance between stemness and differentiation in the root meristem through RNA splicing control.

Plants respond to environmental stresses through controlled stem cell maintenance and meristem activity. One level of gene regulation is RNA alternative splicing. However, the mechanistic link between stress, meristem function and RNA splicing is poorly understood. The MERISTEM-DEFECTIVE (MDF) Arabidopsis gene encodes an SR-related family protein, required for meristem function and leaf vascularization, and is the likely orthologue of the human SART1 and yeast Snu66 splicing factors. MDF is required for the correct splicing and expression of key transcripts associated with root meristem function. We identified RSZ33 and ACC1, both known to regulate cell patterning, as splicing targets required for MDF function in the meristem. MDF expression is modulated by osmotic and cold stress, associated with differential splicing and specific isoform accumulation and shuttling between nucleus and cytosol, and acts in part via a splicing target SR34. We propose a model in which MDF controls splicing in the root meristem to promote stemness and to repress stress response, cell differentiation and cell death pathways.

NAC domain transcription factor gene GhNAC3 confers drought tolerance in plants.

Abiotic stress seriously affects the growth, yield, and fiber quality of cotton. It is of great importance to cultivate drought-resistant and salt-tolerant cotton. NAC (NAM, ATAF1/2, and CUC2) is a plant-specific transcription factor, which is widely involved in the response to abiotic stress. Here, we discovered the GhNAC3 gene isolated from the expression profile of drought stress in cotton and verified its functions in cotton. First, GhNAC3 was strongly induced expression by drought and salt stresses. Gene structure analysis revealed that GhNAC3 had a conserved NAC domain and was homologous to several stress-related NAC transcription factors gene of Arabidopsis. Subcellular localization and transcriptional activation assays revealed that GhNAC3 was a nuclear protein with a C-terminal transcriptional activation domain. Overexpression of GhNAC3 enhanced Arabidopsis tolerance to drought stress with reduced sensitivity to ABA, characterized by increased germination and cotyledon rates under drought stress, and promoted root elongation. VIGS silencing of GhNAC3 reduced cotton tolerance to drought stress as indicated by the low water content of the leaves under drought treatment, significantly faster water loss and lower ABA content in detached leaves, along with the accumulation of more hydrogen peroxide (H2O2) and malondialdehyde (MDA). In conclusion, GhNAC3 plays an important role in the abiotic stress of cotton, which might have great application potential in molecular breeding of cotton varieties with drought resistance.

The GhMAP3K62-GhMKK16-GhMPK32 kinase cascade regulates drought tolerance by activating GhEDT1-mediated ABA accumulation in cotton.

INTRODUCTION: Drought is the principal abiotic stress that severely impacts cotton (Gossypium hirsutum) growth and productivity. Upon sensing drought, plants activate stress-related signal transduction pathways, including ABA signal and mitogen-activated protein kinase (MAPK) cascade. However, as the key components with the fewest members in the MAPK cascade, the function and regulation of GhMKKs need to be elucidated. In addition, the relationship between MAPK module and the ABA core signaling pathway remains incompletely understood. OBJECTIVE: Here we aim to elucidate the molecular mechanism of cotton response to drought, with a focus on mitogen-activated protein kinase (MAPK) cascades activating ABA signaling. METHODS: Biochemical, molecular and genetic analysis were used to study the GhMAP3K62-GhMKK16-GhMPK32-GhEDT1 pathway genes. RESULTS: A nucleus- and membrane-localized MAPK cascade pathway GhMAP3K62-GhMKK16-GhMPK32, which targets and phosphorylates the nuclear-localized transcription factor GhEDT1, to activate downstream GhNCED3 to mediate ABA-induced stomatal closure and drought response was characterized in cotton. Overexpression of GhMKK16 promotes ABA accumulation, and enhances drought tolerance via regulating stomatal closure under drought stress. Conversely, RNAi-mediated knockdown of GhMKK16 expression inhibits ABA accumulation, and reduces drought tolerance. Virus-induced gene silencing (VIGS)-mediated knockdown of either GhMAP3K62, GhMPK32 or GhEDT1 expression represses ABA accumulation and reduces drought tolerance through inhibiting stomatal closure. Expression knockdown of GhMPK32 or GhEDT1 in GhMKK16-overexpressing cotton reinstates ABA content and stomatal opening-dependent drought sensitivity to wild type levels. GhEDT1 could bind to the HD boxes in the promoter of GhNCED3 to activate its expression, resulting in ABA accumulation. We propose that the MAPK cascade GhMAP3K62-GhMKK16-GhMPK32 pathway functions on drought response through ABA-dependent stomatal movement in cotton.

Identification and Functional Analysis of bZIP Genes in Cotton Response to Drought Stress.

The basic leucine zipper (bZIP) transcription factors, which harbor a conserved bZIP domain composed of two regions, a DNA-binding basic region and a Leu Zipper region, operate as important switches of transcription networks in eukaryotes. However, this gene family has not been systematically characterized in cotton (Gossypium hirsutum). Here, we identified 197 bZIP family members in cotton. The chromosome distribution pattern indicates that the GhbZIP genes have undergone 53 genome-wide segmental and 7 tandem duplication events which contribute to the expansion of the cotton bZIP family. Phylogenetic analysis showed that cotton GhbZIP proteins cluster into 13 subfamilies, and homologous protein pairs showed similar characteristics. Inspection of the DNA-binding basic region and leucine repeat heptads within the bZIP domains indicated different DNA-binding site specificities as well as dimerization properties among different groups. Comprehensive expression analysis indicated the most highly and differentially expressed genes in root and leaf that might play significant roles in cotton response to drought stress. GhABF3D was identified as a highly and differentially expressed bZIP family gene in cotton leaf and root under drought stress treatments that likely controls drought stress responses in cotton. These data provide useful information for further functional analysis of the GhbZIP gene family and its potential application in crop improvement.

Silent transcription intervals and translational bursting lead to diverse phenotypic switching.

Gene-expression bimodality, as a potential mechanism generating phenotypic cell diversity, can enhance the survival of cells in a fluctuating environment. Previous studies have shown that intrinsic or extrinsic regulations could induce bimodal gene expressions, but it is unclear whether this bimodality can occur without regulation. Here we develop an interpretable and tractable model, namely a generalized telegraph model (GTM), which considers silent transcription intervals and translational bursting, each being characterized by a general distribution. Using the queuing theory, we derive the analytical expressions of protein distributions, and show that non-exponential inactive times and translational bursting can lead to two peaks of the protein distribution away from the origin, which are different from those occurring in classical telegraph models. We also find that both silent-interval noise and translational burst-size noise can amplify gene-expression noise and induce diverse dynamic expression patterns. Our results not only provide an alternative mechanism of phenotypic switching but also could be used in explaining the bimodal phenomenon in experimental observations.

Single-cell RNA-seq reveals fate determination control of an individual fibre cell initiation in cotton (Gossypium hirsutum).

Cotton fibre is a unicellular seed trichome, and lint fibre initials per seed as a factor determines fibre yield. However, the mechanisms controlling fibre initiation from ovule epidermis are not understood well enough. Here, with single-cell RNA sequencing (scRNA-seq), a total of 14 535 cells were identified from cotton ovule outer integument of Xu142_LF line at four developmental stages (1.5, 1, 0.5 days before anthesis and the day of anthesis). Three major cell types, fibre, non-fibre epidermis and outer pigment layer were identified and then verified by RNA in situ hybridization. A comparative analysis on scRNA-seq data between Xu142 and its fibreless mutant Xu142 fl further confirmed fibre cluster definition. The developmental trajectory of fibre cell was reconstructed, and fibre cell was identified differentiated at 1 day before anthesis. Gene regulatory networks at four stages revealed the spatiotemporal pattern of core transcription factors, and MYB25-like and HOX3 were demonstrated played key roles as commanders in fibre differentiation and tip-biased diffuse growth respectively. A model for early development of a single fibre cell was proposed here, which sheds light on further deciphering mechanism of plant trichome and the improvement of cotton fibre yield.

Transcriptome and metabolome profiling of interspecific CSSLs reveals general and specific mechanisms of drought resistance in cotton.

In order to understand the molecular mechanism of cotton's response to drought during the flowering and boll stage, transcriptomics and metabolomics were carried out for two introgression lines (drought-tolerant line: T307; drought-sensitive line: S48) which were screened from Gossypium hirsutum cv. 'Emian22' with some gene fragments imported from Gossypium barbadense acc. 3-79, under drought stress by withdrawing water at flowering and boll stage. Results showed that the basic drought response in cotton included a series of broad-spectrum responses, such as amino acid synthesis, hormone (abscisic acid, ABA) signal transduction, and mitogen-activated protein kinases signal transduction pathway, which activated in both drought-tolerant and drought-sensitive lines. However, the difference of their imported fragments and diminished sequences triggers endoplasmic reticulum (ER) protein processing, photosynthetic-related pathways (in leaves), and membrane solute transport (in roots) in drought-tolerant line T307, while these are missed or not activated in drought-sensitive line S48, reflecting the different drought tolerance of the two genotypes. Virus-induced gene silencing assay of drought-tolerant differentially expressed heat shock protein (HSP) genes (mainly in leaf) and ATP-binding cassette (ABC) transporter genes (mainly in roots) indicated that those genes play important role in cotton drought tolerant. Combined analysis of transcriptomics and metabolomics highlighted the important roles of ER-stress-related HSP genes and root-specific ABC transporter genes in plants drought tolerance. These results provide new insights into the molecular mechanisms underlying the drought stress adaptation in cotton.

GhTCE1-GhTCEE1 dimers regulate transcriptional reprogramming during wound-induced callus formation in cotton.

Wounded plant cells can form callus to seal the wound site. Alternatively, wounding can cause adventitious organogenesis or somatic embryogenesis. These distinct developmental pathways require specific cell fate decisions. Here, we identify GhTCE1, a basic helix-loop-helix family transcription factor, and its interacting partners as a central regulatory module of early cell fate transition during in vitro dedifferentiation of cotton (Gossypium hirsutum). RNAi- or CRISPR/Cas9-mediated loss of GhTCE1 function resulted in excessive accumulation of reactive oxygen species (ROS), arrested callus cell elongation, and increased adventitious organogenesis. In contrast, GhTCE1-overexpressing tissues underwent callus cell growth, but organogenesis was repressed. Transcriptome analysis revealed that several pathways depend on proper regulation of GhTCE1 expression, including lipid transfer pathway components, ROS homeostasis, and cell expansion. GhTCE1 bound to the promoters of the target genes GhLTP2 and GhLTP3, activating their expression synergistically, and the heterodimer TCE1-TCEE1 enhances this activity. GhLTP2- and GhLTP3-deficient tissues accumulated ROS and had arrested callus cell elongation, which was restored by ROS scavengers. These results reveal a unique regulatory network involving ROS and lipid transfer proteins, which act as potential ROS scavengers. This network acts as a switch between unorganized callus growth and organized development during in vitro dedifferentiation of cotton cells.

Kinetic characteristics of transcriptional bursting in a complex gene model with cyclic promoter structure.

While transcription often occurs in a bursty manner, various possible regulations can lead to complex promoter patterns such as promoter cycles, giving rise to an important question: How do promoter kinetics shape transcriptional bursting kinetics? Here we introduce and analyze a general model of the promoter cycle consisting of multi-OFF states and multi-ON states, focusing on the effects of multi-ON mechanisms on transcriptional bursting kinetics. The derived analytical results indicate that burst size follows a mixed geometric distribution rather than a single geometric distribution assumed in previous studies, and ON and OFF times obey their own mixed exponential distributions. In addition, we find that the multi-ON mechanism can lead to bimodal burst-size distribution, antagonistic timing of ON and OFF, and diverse burst frequencies, each further contributing to cell-to-cell variability in the mRNA expression level. These results not only reveal essential features of transcriptional bursting kinetics patterns shaped by multi-state mechanisms but also can be used to the inferences of transcriptional bursting kinetics and promoter structure based on experimental data.