Biotinylated Tn5 transposase-mediated CUT&Tag efficiently profiles transcription factor-DNA interactions in plants.

In contrast to CUT&Tag approaches for profiling bulk histone modifications, current CUT&Tag methods for analysing specific transcription factor (TF)-DNA interactions remain technically challenging due to TFs having relatively low abundance. Moreover, an efficient CUT&Tag strategy for plant TFs is not yet available. Here, we first applied biotinylated Tn5 transposase-mediated CUT&Tag (B-CUT&Tag) to produce high-quality libraries for interrogating TF-DNA interactions. B-CUT&Tag combines streptavidin-biotin-based DNA purification with routine CUT&Tag, optimizing the removal of large amounts of intact chromatin not targeted by specific TFs. The biotinylated chromatin fragments are then purified for construction of deep sequencing libraries or qPCR analysis. We applied B-CUT&Tag to probe genome-wide DNA targets of Squamosa promoter-binding-like protein 9 (SPL9), a well-established TF in Arabidopsis; the resulting profiles were efficient and consistent in demonstrating its well-established target genes in juvenile-adult transition/flowering, trichome development, flavonoid biosynthesis, wax synthesis and branching. Interestingly, our results indicate functions of AtSPL9 in modulating growth-defence trade-offs. In addition, we established a method for applying qPCR after CUT&Tag (B-CUT&Tag-qPCR) and successfully validated the binding of SPL9 in Arabidopsis and PHR2 in rice. Our study thus provides a convenient and highly efficient CUT&Tag strategy for profiling TF-chromatin interactions that is widely applicable to the annotation of cis-regulatory elements for crop improvement.

Shear Stress Rescued the Neuronal Impairment Induced by Global Cerebral Ischemia Reperfusion via Activating PECAM-1-eNOS-NO Pathway.

Microvessel hypoperfusion following ischemic stress resulted in a decreased shear stress of brain microvascular endothelial cells (BMECs) and contributed to abnormal expression of PECAM-1 after global cerebral ischemia/reperfusion (I/R) injury. Here, we identified novel pathophysiologic and rehabilitative procedures specific to shear stress in microvascular endothelial cells in response to global cerebral I/R injury. We found that the decrease in cerebral blood flow of gerbils after global cerebral I/R injury reduces shear stress, and the abnormal change in shear stress leads to microvascular endothelial cell and neuron damage. Nevertheless, suitable high levels of shear stress contribute to rescuing the dysfunction and malformation of BMECs via regulating the PECAM-1-eNOS-NO pathway to enhance nitric oxide release, decrease the expression of caspase-3 to reduce apoptosis, and improve the shear-adaptability of endothelial cells, thereby playing a protective role in the gerbil brain.

The miR319-Targeted GhTCP4 Promotes the Transition from Cell Elongation to Wall Thickening in Cotton Fiber.

Plant cell growth involves a complex interplay among cell-wall expansion, biosynthesis, and, in specific tissues, secondary cell wall (SCW) deposition, yet the coordination of these processes remains elusive. Cotton fiber cells are developmentally synchronous, highly elongated, and contain nearly pure cellulose when mature. Here, we report that the transcription factor GhTCP4 plays an important role in balancing cotton fiber cell elongation and wall synthesis. During fiber development the expression of miR319 declines while GhTCP4 transcript levels increase, with high levels of the latter promoting SCW deposition. GhTCP4 interacts with a homeobox-containing factor, GhHOX3, and repressing its transcriptional activity. GhTCP4 and GhHOX3 function antagonistically to regulate cell elongation, thereby establishing temporal control of fiber cell transition to the SCW stage. We found that overexpression of GhTCP4A upregulated and accelerated activation of the SCW biosynthetic pathway in fiber cells, as revealed by transcriptome and promoter activity analyses, resulting in shorter fibers with varied lengths and thicker walls. In contrast, GhTCP4 downregulation led to slightly longer fibers and thinner cell walls. The GhHOX3-GhTCP4 complex may represent a general mechanism of cellular development in plants since both are conserved factors in many species, thus providing us a potential molecular tool for the design of fiber traits.

The HD-Zip IV gene GaHOX1 from cotton is a functional homologue of the Arabidopsis GLABRA2.

Most of the plant homeodomain-containing proteins play important roles in organ patterning and development, and Arabidopsis GLABRA2 (GL2), a member of the class IV homeodomain-leucine zipper (HD-ZIP) proteins, is a trichome and non-root hair cell regulator. Here we report the analysis of two cotton homeodomain-containing proteins, GaHOX1 and GaHOX2, isolated from the diploid cotton Gossypium arboreum. Both GaHOX1 and GaHOX2 belong to the class IV HD-ZIP family. When expressed under the control of the GL2 promoter, GaHOX1 rescued trichome development of an Arabidopsis glabrous mutant of gl2-2 (SALK_130213), whereas GaHOX2 did not. On the other hand, expression of GaHOX1 with a Cauliflower mosaic virus (CaMV) 35S promoter in the wild-type Arabidopsis plants suppressed the trichome development just as the GL2 ectopic expression. Expression analysis by Northern, RT-PCR and in situ hybridization indicated that GaHOX1 is predominantly expressed in cotton fiber cells at early developmental stages, consistent with its putative role in regulating cotton fiber development, while GaHOX2 is expressed in both fiber and other ovular tissues, including outer and inner integuments. Our results suggest that GaHOX1 is a functional homolog of GL2 in plant trichome development.

Control of cotton fibre elongation by a homeodomain transcription factor GhHOX3.

Cotton fibres are unusually long, single-celled epidermal seed trichomes and a model for plant cell growth, but little is known about the regulation of fibre cell elongation. Here we report that a homeodomain-leucine zipper (HD-ZIP) transcription factor, GhHOX3, controls cotton fibre elongation. GhHOX3 genes are localized to the 12th homoeologous chromosome set of allotetraploid cotton cultivars, associated with quantitative trait loci (QTLs) for fibre length. Silencing of GhHOX3 greatly reduces (>80%) fibre length, whereas its overexpression leads to longer fibre. Combined transcriptomic and biochemical analyses identify target genes of GhHOX3 that also contain the L1-box cis-element, including two cell wall loosening protein genes GhRDL1 and GhEXPA1. GhHOX3 interacts with GhHD1, another homeodomain protein, resulting in enhanced transcriptional activity, and with cotton DELLA, GhSLR1, repressor of the growth hormone gibberellin (GA). GhSLR1 interferes with the GhHOX3-GhHD1 interaction and represses target gene transcription. Our results uncover a novel mechanism whereby a homeodomain protein transduces GA signal to promote fibre cell elongation.