Transcriptomic Analysis Reveals the Key Role of Histone Deacetylation-mediated Phytohormones Interaction in Fiber Initiation of Cotton

Background: Histone deacetylation is one of the most important epigenetic modifications and plays diverse roles in plant development. However, the detailed functions and mechanisms of histone deacetylation in fiber development of cotton are still unclear. HDAC inhibitors (HDACi) have been used commonly to study the molecular mechanism underlying histone deacetylation or to facilitate disease therapy in humans through hindering the histone deacetylase catalytic activity. Trichostatin A (TSA) - the most widely used HDACi has been used to determine the role of histone deacetylation on different developmental stages of plants. Results: Here, exogenous TSA was applied in the fiber initiation and elongation in vitro, and the results demonstrated the crucial role of histone deacetylation in fiber initiation regulation. Therefore, we made a transcriptomic analysis to reveal the underlying mechanisms. Through RNA-Seq analysis, the differentially expressed genes were mostly enriched in plant hormone signal transduction,phenylpropanoid biosynthesis, photosynthesis, and carbon metabolism pathways, suggesting the potential role of phytohormone, phenylpropanoid metabolism, and energy metabolism downstream of histone deacetylation in fiber initiation. The phytohormone signal transduction pathways harbor the most differentially expressed genes. Deeper studies showed that some genes promoting auxin, Gibberellic Acid (GA), and Salicylic Acid (SA) signaling were down-regulated, while some genes facilitating Abscisic Acid (ABA) and inhibiting Jasmonic Acid (JA) singling were up-regulated after the TSA treatments. Conclusions: Collectively, we established a model, in which histone deacetylation can regulate some key genes involved in different phytohormone pathways, consequently, promoting the auxin, GA, JA and SA signaling, whereas, repressing the ABA signaling to improve the fiber cell initiation; besides that the genes associated with energy metabolism, phenylpropanoid, and glutathione metabolism were also involved in. The above results provided novel clues to illuminate the underlying mechanisms of epigenetic modifications as well as interactions of different phytohormones in fiber cell differentiation, which is also very valuable for molecular breeding of higher quality cotton.

Microstructure Transformation In Palm-Fiber Cell Wall And Its Influence On the Fiber's Mechanical Properties During Alkali Treatment

In this study, we examined the microstructure transformation of palm fiber and the influence of this transformation on the fiber mechanical properties during alkali treatment. The fibers were treated with different concentrations of NaOH to study the change rules of the microstructure and the tensile properties. FT-IR microspectroscopic imaging and confocal laser scanning microscopy were adopted to observe microstructure transformation during alkali treatment. Research results showed that the hemicellulose and lignin in the fiber cell wall were removed by alkali treatment, leading to a rearrangement of cellulose chains. The tensile properties palm fibers were significantly improved because of crystallinity alterations in the cell walls after alkali treatment. This study might provide a basis for palm fiber’s high-value utilization in the field of materials.

Eph-ephrin Signaling Affects Eye Lens Fiber Cell Intracellular Voltage and Membrane Conductance

The avascular eye lens generates its own microcirculation that is required for maintaining lifelong lens transparency. The microcirculation relies on sodium ion flux, an extensive network of gap junction (GJ) plaques between lens fiber cells and transmembrane water channels. Disruption of connexin proteins, the building blocks of GJs, or aquaporins, which make up water and adhesion channels, lead to lens opacification or cataracts. Recent studies have revealed that disruption of Eph-ephrin signaling, in particular the receptor EphA2 and the ligand ephrin-A5, in humans and mice lead to congenital and age-related cataracts. We investigated whether changes in lens transparency in EphA2 or ephrin-A5 knockout (–/–) mice is related to changes in GJ coupling and lens fluid and ion homeostasis. Immunostaining revealed changes in connexin 50 (Cx50) subcellular localization in EphA2–/– peripheral lens fibers and alteration in aquaporin 0 (Aqp0) staining patterns in ephrin-A5–/– and EphA2–/– inner mature fiber cells. Surprisingly, there was no obvious change in GJ coupling in knockout lenses. However, there were changes in fiber cell membrane conductance and intracellular voltage in knockout lenses from 3-month-old mice. These knockout lenses displayed decreased conductance of mature fiber membranes and were hyperpolarized compared to control lenses. This is the first demonstration that the membrane conductance of lens fibers can be regulated. Together these data suggest that EphA2 may be needed for normal Cx50 localization to the cell membrane and that conductance of lens fiber cells requires normal Eph-ephrin signaling and water channel localization.

Comparative Metabolomics Analysis Reveals Sterols and Sphingolipids Play a Role in Cotton Fiber Cell Initiation

Cotton fiber is a seed trichome that protrudes from the outer epidermis of cotton ovule on the day of anthesis (0 day past anthesis, 0 DPA). The initial number and timing of fiber cells are closely related to fiber yield and quality. However, the mechanism underlying fiber initiation is still unclear. Here, we detected and compared the contents and compositions of sphingolipids and sterols in 0 DPA ovules of Xuzhou142 lintless-fuzzless mutants (Xufl) and Xinxiangxiaoji lintless-fuzzless mutants (Xinfl) and upland cotton wild-type Xuzhou142 (XuFL). Nine classes of sphingolipids and sixty-six sphingolipid molecular species were detected in wild-type and mutants. Compared with the wild type, the contents of Sphingosine-1-phosphate (S1P), Sphingosine (Sph), Glucosylceramide (GluCer), and Glycosyl-inositol-phospho-ceramides (GIPC) were decreased in the mutants, while the contents of Ceramide (Cer) were increased. Detail, the contents of two Cer molecular species, d18:1/22:0 and d18:1/24:0, and two Phyto-Cer molecular species, t18:0/22:0 and t18:0/h22:1 were significantly increased, while the contents of all GluCer and GIPC molecular species were decreased. Consistent with this result, the expression levels of seven genes involved in GluCer and GIPC synthesis were decreased in the mutants. Furthermore, exogenous application of a specific inhibitor of GluCer synthase, PDMP (1-phenyl-2-decanoylamino-3-morpholino-1-propanol), in ovule culture system, significantly inhibited the initiation of cotton fiber cells. In addition, five sterols and four sterol esters were detected in wild-type and mutant ovules. Compared with the wild type, the contents of total sterol were not significantly changed. While the contents of stigmasterol and campesterol were significantly increased, the contents of cholesterol were significantly decreased, and the contents of total sterol esters were significantly increased. In particular, the contents of campesterol esters and stigmasterol esters increased significantly in the two mutants. Consistently, the expression levels of some sterol synthase genes and sterol ester synthase genes were also changed in the two mutants. These results suggested that sphingolipids and sterols might have some roles in the initiation of fiber cells. Our results provided a novel insight into the regulatory mechanism of fiber cell initiation.

Mutation of the EPHA2 Tyrosine-Kinase Domain Dysregulates Cell Pattern Formation and Cytoskeletal Gene Expression in the Lens

Genetic variations in ephrin type-A receptor 2 (EPHA2) have been associated with inherited and age-related forms of cataract in humans. Here, we have characterized the eye lens phenotype and transcript profile of germline Epha2 knock-in mutant mice homozygous for either a missense variant associated with age-related cataract in humans (Epha2-Q722) or a novel insertion-deletion mutation (Epha2-indel722) that were both located within the tyrosine-kinase domain of EPHA2. Confocal imaging of ex vivo lenses from Epha2-indel722 mice on a fluorescent reporter background revealed misalignment of epithelial-to-fiber cell meridional-rows at the lens equator and severe disturbance of Y-suture formation at the lens poles, whereas Epha2-Q722 lenses displayed mild disturbance of posterior sutures. Immunofluorescent labeling showed that EPHA2 was localized to radial columns of hexagonal fiber cell membranes in Epha2-Q722 lenses, whereas Epha2-indel722 lenses displayed disorganized radial cell columns and cytoplasmic retention of EPHA2. Immunoprecipitation/blotting studies indicated that EPHA2 formed strong complexes with Src kinase and was mostly serine phosphorylated in the lens. RNA sequencing analysis revealed differential expression of several cytoskeleton-associated genes in Epha2-mutant and Epha2-null lenses including shared downregulation of Lgsn and Clic5. Collectively, our data suggest that mutations within the tyrosine-kinase domain of EPHA2 result in lens cell patterning defects and dysregulated expression of several cytoskeleton-associated proteins.

Sphingolipid Profile during Cotton Fiber Growth Revealed That a Phytoceramide Containing Hydroxylated and Saturated VLCFA Is Important for Fiber Cell Elongation

Cotton fiber is a single-celled seed trichrome that arises from the epidermis of the ovule’s outer integument. The fiber cell displays high polar expansion and thickens but not is disrupted by cell division. Therefore, it is an ideal model for studying the growth and development of plant cells. Sphingolipids are important components of membranes and are also active molecules in cells. However, the sphingolipid profile during fiber growth and the differences in sphingolipid metabolism at different developmental stages are still unclear. In this study, we detected that there were 6 classes and 95 molecular species of sphingolipids in cotton fibers by ultrahigh performance liquid chromatography-MS/MS (UHPLC-MS/MS). Among these, the phytoceramides (PhytoCer) contained the most molecular species, and the PhytoCer content was highest, while that of sphingosine-1-phosphate (S1P) was the lowest. The content of PhytoCer, phytoceramides with hydroxylated fatty acyls (PhytoCer-OHFA), phyto-glucosylceramides (Phyto-GluCer), and glycosyl-inositol-phospho-ceramides (GIPC) was higher than that of other classes in fiber cells. With the development of fiber cells, phytosphingosine-1-phosphate (t-S1P) and PhytoCer changed greatly. The sphingolipid molecular species Ceramide (Cer) d18:1/26:1, PhytoCer t18:1/26:0, PhytoCer t18:0/26:0, PhytoCer t18:1/h20:0, PhytoCer t18:1/h26:0, PhytoCer t18:0/h26:0, and GIPC t18:0/h16:0 were significantly enriched in 10-DPA fiber cells while Cer d18:1/20:0, Cer d18:1/22:0, and GIPC t18:0/h18:0 were significantly enriched in 20-DPA fiber cells, indicating that unsaturated PhytoCer containing hydroxylated and saturated very long chain fatty acids (VLCFA) play some role in fiber cell elongation. Consistent with the content analysis results, the related genes involved in long chain base (LCB) hydroxylation and unsaturation as well as VLCFA synthesis and hydroxylation were highly expressed in rapidly elongating fiber cells. Furthermore, the exogenous application of a potent inhibitor of serine palmitoyltransferase, myriocin, severely blocked fiber cell elongation, and the exogenous application of sphingosine antagonized the inhibition of myriocin for fiber elongation. Taking these points together, we concluded that sphingolipids play crucial roles in fiber cell elongation and SCW deposition. This provides a new perspective for further studies on the regulatory mechanism of the growth and development of cotton fiber cells.

A functional map of genomic HIF1α-DNA complexes in the eye lens revealed through multiomics analysis

Background During eye lens development the embryonic vasculature regresses leaving the lens without a direct oxygen source. Both embryonically and throughout adult life, the lens contains a decreasing oxygen gradient from the surface to the core that parallels the natural differentiation of immature surface epithelial cells into mature core transparent fiber cells. These properties of the lens suggest a potential role for hypoxia and the master regulator of the hypoxic response, hypoxia-inducible transcription factor 1 (HIF1), in the regulation of genes required for lens fiber cell differentiation, structure and transparency. Here, we employed a multiomics approach combining CUT&RUN, RNA-seq and ATACseq analysis to establish the genomic complement of lens HIF1α binding sites, genes activated or repressed by HIF1α and the chromatin states of HIF1α-regulated genes. Results CUT&RUN analysis revealed 8375 HIF1α-DNA binding complexes in the chick lens genome. One thousand one hundred ninety HIF1α-DNA binding complexes were significantly clustered within chromatin accessible regions (χ2 test p < 1 × 10− 55) identified by ATACseq. Formation of the identified HIF1α-DNA complexes paralleled the activation or repression of 526 genes, 116 of which contained HIF1α binding sites within 10kB of the transcription start sites. Some of the identified HIF1α genes have previously established lens functions while others have novel functions never before examined in the lens. GO and pathway analysis of these genes implicate HIF1α in the control of a wide-variety of cellular pathways potentially critical for lens fiber cell formation, structure and function including glycolysis, cell cycle regulation, chromatin remodeling, Notch and Wnt signaling, differentiation, development, and transparency. Conclusions These data establish the first functional map of genomic HIF1α-DNA complexes in the eye lens. They identify HIF1α as an important regulator of a wide-variety of genes previously shown to be critical for lens formation and function and they reveal a requirement for HIF1α in the regulation of a wide-variety of genes not yet examined for lens function. They support a requirement for HIF1α in lens fiber cell formation, structure and function and they provide a basis for understanding the potential roles and requirements for HIF1α in the development, structure and function of more complex tissues.