The transmembrane protein LbRSG from the recretohalophyte Limonium bicolor enhances salt gland development and salt tolerance.

Salt glands are the unique epidermal structures present in recretohalophytes, plants that actively excrete excess Na+ by salt secretory structures to avoid salt damage. Here, we describe a transmembrane protein that localizes to the plasma membrane of the recretohalophyte Limonium bicolor. As virus-induced gene silencing of the corresponding gene LbRSG in L. bicolor decreased the number of salt glands, we named the gene Reduced Salt Gland. We detected LbRSG transcripts in salt glands by in situ hybridization and transient transformation. Overexpression and silencing of LbRSG in L. bicolor pointed to a positive role in salt gland development and salt secretion by interacting with Lb3G16832. Heterologous LbRSG expression in Arabidopsis enhanced salt tolerance during germination and the seedling stage by alleviating NaCl-induced ion stress and osmotic stress after replacing or deleting the (highly) negatively charged region of extramembranous loop. After screened by immunoprecipitation-mass spectrometry and verified using yeast two-hybrid, PGK1 and BGLU18 were proposed to interact with LbRSG to strengthen salt tolerance. Therefore, we identified (highly) negatively charged regions in the extramembrane loop that may play an essential role in salt tolerance, offering hints about LbRSG function and its potential to confer salt resistance.

Structural basis of the human negative elongation factor NELF-B/C/E ternary complex.

Negative elongation factor (NELF) is a four-subunit transcription elongation factor that mainly functions in maintaining the paused state of RNA polymerase II in eukaryotes. Upon binding to Pol II, NELF works synergistically with DRB sensitivity-inducing factor (DSIF) and inhibits transcription elongation of Pol II, which subsequently retains a stably paused state 20-60 base pairs downstream of the promoter. The promoter-proximal pausing of Pol II caused by NELF is a general mechanism of transcriptional regulation for most signal-responsive genes. To date, structural studies have significantly advanced our understanding of the molecular mechanisms of NELF. However, a high quality structural model clarifying the interaction details of this complex is still lacking. In this study, we solved the high resolution crystal structure of the NELF-B/C/E ternary complex. We observed detailed interactions between subunits and identified residues important for the association between NELF-B and NELF-E. Our work presents a precise model of the NELF complex, which will facilitate our understanding of its in vivo function.

Halostachys caspica pathogenesis-related protein 10 acts as a cytokinin reservoir to regulate plant growth and development.

Pathogenesis-related class 10 (PR-10) proteins play a role in plant growth and development, but the underlying molecular mechanisms are unclear. Here, we isolated a salt-induced PR-10 gene from the halophyte Halostachys caspica and named it HcPR10. HcPR10 was constitutively expressed during development and HcPR10 localized to the nucleus and cytoplasm. HcPR10-mediated phenotypes including bolting, earlier flowering, increased branch number and siliques per plant are highly correlated with increased cytokinin levels in transgenic Arabidopsis. Meanwhile, increased levels of cytokinin in plants is temporally correlated with HcPR10 expression patterns. Although the expression of cytokinin biosynthesis genes validated was not upregulated, cytokinin-related genes including chloroplast-related genes, cytokinin metabolism and cytokinin responses genes and flowering-related genes were significantly upregulated in the transgenic Arabidopsis compared to the wild type by transcriptome deep sequencing. Analysis of the crystal structure of HcPR10 revealed a trans-zeatin riboside (a type of cytokinin) located deep in its cavity, with a conserved conformation and protein-ligand interactions, supporting HcRP10 acts as a cytokinin reservoir. Moreover, HcPR10 in Halostachys caspica predominantly accumulated in vascular tissue, the site of long-distance translocation of plant hormones. Collectively, we draw that HcPR10 as a cytokinin reservoir induces cytokinin-related signal transduction in plants, thereby promoting plant growth and development. These findings could provide intriguing insights into the role of HcPR10 proteins in phytohormone regulation in plants and advance our understanding of cytokinin-mediated plant development and could facilitate the breeding of transgenic crops with earlier mature, higher yielding agronomic traits.

Structural Basis of the Transcriptional Elongation Factor Paf1 Core Complex from Saccharomyces eubayanus.

The multicomponent polymerase associated factor 1 (Paf1) complex (PAF1C) is an important transcription elongation factor that upregulates RNA polymerase II-mediated genome-wide transcription. PAF1C can regulate transcription through direct association with the polymerase or by impacting the chromatin structure epigenetically. In recent years, significant progress has been made in understanding the molecular mechanisms of PAF1C. However, high-resolution structures that can clarify the interaction details among the components of the complex are still needed. In this study, we evaluated the structural core of the yeast PAF1C containing the four components Ctr9, Paf1, Cdc73 and Rtf1 at high resolution. We observed the interaction details among these components. In particular, we identified a new binding surface of Rtf1 on PAF1C and found that the C-terminal sequence of Rtf1 dramatically changed during evolution, which may account for its different binding affinities to PAF1C among species. Our work presents a precise model of PAF1C, which will facilitate our understanding of the molecular mechanism and the in vivo function of the yeast PAF1C.

The histone acetyltransferase KAT6A is recruited to unmethylated CpG islands via a DNA binding winged helix domain.

The lysine acetyltransferase KAT6A (MOZ, MYST3) belongs to the MYST family of chromatin regulators, facilitating histone acetylation. Dysregulation of KAT6A has been implicated in developmental syndromes and the onset of acute myeloid leukemia (AML). Previous work suggests that KAT6A is recruited to its genomic targets by a combinatorial function of histone binding PHD fingers, transcription factors and chromatin binding interaction partners. Here, we demonstrate that a winged helix (WH) domain at the very N-terminus of KAT6A specifically interacts with unmethylated CpG motifs. This DNA binding function leads to the association of KAT6A with unmethylated CpG islands (CGIs) genome-wide. Mutation of the essential amino acids for DNA binding completely abrogates the enrichment of KAT6A at CGIs. In contrast, deletion of a second WH domain or the histone tail binding PHD fingers only subtly influences the binding of KAT6A to CGIs. Overexpression of a KAT6A WH1 mutant has a dominant negative effect on H3K9 histone acetylation, which is comparable to the effects upon overexpression of a KAT6A HAT domain mutant. Taken together, our work revealed a previously unrecognized chromatin recruitment mechanism of KAT6A, offering a new perspective on the role of KAT6A in gene regulation and human diseases.

The SAM domain-containing protein 1 (SAMD1) acts as a repressive chromatin regulator at unmethylated CpG islands.

CpG islands (CGIs) are key regulatory DNA elements at most promoters, but how they influence the chromatin status and transcription remains elusive. Here, we identify and characterize SAMD1 (SAM domain-containing protein 1) as an unmethylated CGI-binding protein. SAMD1 has an atypical winged-helix domain that directly recognizes unmethylated CpG-containing DNA via simultaneous interactions with both the major and the minor groove. The SAM domain interacts with L3MBTL3, but it can also homopolymerize into a closed pentameric ring. At a genome-wide level, SAMD1 localizes to H3K4me3-decorated CGIs, where it acts as a repressor. SAMD1 tethers L3MBTL3 to chromatin and interacts with the KDM1A histone demethylase complex to modulate H3K4me2 and H3K4me3 levels at CGIs, thereby providing a mechanism for SAMD1-mediated transcriptional repression. The absence of SAMD1 impairs ES cell differentiation processes, leading to misregulation of key biological pathways. Together, our work establishes SAMD1 as a newly identified chromatin regulator acting at unmethylated CGIs.

An OFF-ON-OFF type fluorescent probe based on a naphthalene derivative for Al3+ and F- ions and its biological application.

A novel fluorescent probe-based naphthalene Schiff, 1-(C2-glucosyl-ylimino-methyl)-naphthalene-2-ol (L) was synthesized by coupling d-glucosamine hydrochloride with 2-hydroxy-1-naphthaldehyde. It exhibited excellent selectivity and highly sensitivity for Al3+ in ethanol with a strong fluorescence response, while other common metal ions such as Pb2+ , Mg2+ , Cu2+ , Co2+ , Ni2+ , Cd2+ , Fe2+ , Mn2+ , Hg2+ , Li+ , Na+ , K+ , Fe3+ , Cr3+ , Zn2+ , Ag+ , Ba2+ and Ca2+ did not cause the same fluorescence response. The probe selectively bound Al3+ with a binding constant (Ka ) of 5.748 × 103  M-1 and a lowest detection limit (LOD) of 4.08 nM. Moreover, the study found that the fluorescence of the L - Al3+ complex could be quenched after addition of F- in the same medium, while other anions, including Cl- , Br- , I- , NO2- , NO3- , ClO4- , CO32- , HCO3- , SO42- , HSO4- , CH3 COO- , PO43- , HPO42- , S2- and S2 O32- had nearly no influence on probe behaviour. Binding of the [L - Al3+ ] complex to a F- anion was established by different fluorescence titration studies, with a detection limit of 3.2 nM in ethanol. The fluorescent probe was also successfully applied in the imaging detection of Al3+ and F- in living cells.