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A major obstacle for orchid (Orchidaceae) breeding and production is a long juvenile phase before orchid reproductive development. The molecular basis for prolonged vegetative growth in orchids remains largely unclear despite many efforts to clarify the relevant mechanisms. In this study, we report functional characterization of Dendrobium Orchid TERMINAL FLOWER1 (DOTFL1), an ortholog of TFL1 in Arabidopsis (Arabidopsis thaliana), from the orchid Dendrobium Chao Praya Smile. DOTFL1 is highly expressed in pseudobulbs and the shoot apical meristem (SAM) before and during the floral transition, but is downregulated in inflorescence apices and open flowers. Ectopic expression of DOTFL1 rescues the early-flowering and terminal-flower phenotypes of tfl1-20 in Arabidopsis. Overexpression of DOTFL1 in Dendrobium orchids delays flowering and produces defective inflorescence meristems and flowers with vegetative traits, whereas knockdown of DOTFL1 accelerates flowering and perturbs the maintenance of the inflorescence meristem. Notably, DOTFL1 suppresses orchid flowering and associated pseudobulb formation during the floral transition. We further reveal that two orchid MADS-box transcription factors, Dendrobium Orchid SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (DOSOC1) and AGAMOUS-LIKE 24 (DOAGL24), could interact with each other and bind to the CArG-box motif at DOTFL1, implying a regulatory hierarchy similar to their counterparts in Arabidopsis. Taken together, our findings suggest that DOTFL1 promotes vegetative growth, modulates successive developmental events required for reproductive success in Dendrobium orchids, and may have evolved with a previously unknown role in controlling pseudobulb formation in the Orchidaceae family.

PURPOSE To quantitatively analyze the impact of intrahepatic venovenous shunt (IHVS) on hepatic venous pressure gradient (HVPG) measurement. MATERIALS AND METHODS From 2015 to 2019, 222 HVPG measurements performed during transjugular intrahepatic portosystemic shunt creation were eligible for this study. Digital subtraction angiography (DSA) software color-coded each pixel of a two-dimensional DSA series by time-intensity curve to classify IHVS. Different degrees of IHVS were found in 36.5% of patients (81/222). Mild IHVS was found in 10.8% of patients (24/222), moderate IHVS was found in 10.8% of patients (24/222), and severe IVHS was found in 14.9% of patients (33/222). RESULTS Mean wedged hepatic vein pressure (WHVP) and HVPG were significantly lower in patients with IHVS compared with patients without IHVS (WHVP: 17.78 mm Hg ± 7.00 vs 24.89 mm Hg ± 8.69, P = .001; HVPG: 11.93 mm Hg ± 5.76 vs 18.6 mm Hg ± 6.85, P < .001). Mild IHVS had little effect on WHVP and HVPG. Mean WHVP and HVPG were 11 mm Hg lower in patients with moderate IHVS (WHVP: 20.38 mm Hg ± 8.38 vs 31.5 mm Hg ± 9.39, P = .026; HVPG: 13.88 mm Hg ± 6.33 vs 25.00 mm Hg ± 9.81, P < .001) and 15 mm Hg lower in patients with severe IHVS (WHVP: 13.45 mm Hg ± 5.28 vs 28.64 mm Hg ± 6.38, P = .017; HVPG: 8.27 mm Hg ± 3.85 vs 23.45 mm Hg ± 6.95, P < .001) than mean portal vein pressure and portal vein gradient. CONCLUSIONS For patients with moderate or severe IHVS, HVPG might greatly underestimate the actual value of portal vein pressure, and the portal vein should be catheterized to measure portal pressure.

Background: Hepatocellular carcinoma (HCC) is an aggressive primary hepatic cancer with high malignancy and poor prognosis. Long noncoding RNA HOTAIR has been classified as an oncogene to accelerat...

The prevalence of chronic kidney disease (CKD) is rapidly increasing over the last few decades, owing to the global increase in diabetes, and cardiovascular diseases. Dialysis greatly compromises the life quality of patients, while demand for transplantable kidney cannot be met, underscoring the need to develop novel therapeutic approaches to stop or reverse CKD progression. Our understanding of kidney disease is primarily derived from studies using animal models and cell culture. While cross-species differences made it challenging to fully translate findings from animal models into clinical practice, primary patient cells quickly lose the original phenotypes during in vitro culture. Over the last decade, remarkable achievements have been made for generating 3-dimensional (3D) miniature organs (organoids) by exposing stem cells to culture conditions that mimic the signaling cues required for the development of a particular organ or tissue. 3D kidney organoids have been successfully generated from different types of source cells, including human pluripotent stem cells (hPSCs), adult/fetal renal tissues, and kidney cancer biopsy. Alongside gene editing tools, hPSC-derived kidney organoids are being harnessed to model genetic kidney diseases. In comparison, adult kidney-derived tubuloids and kidney cancer-derived tumoroids are still in their infancy. Herein, we first summarize the currently available kidney organoid models. Next, we discuss recent advances in kidney disease modelling using organoid models. Finally, we consider the major challenges that have hindered the application of kidney organoids in disease modelling and drug evaluation and propose prospective solutions. Ministry of Education (MOE) Nanyang Technological University National Medical Research Council (NMRC) Published version This work is supported by MOE Grants (MOE2019-T2-1-072, MOE-T2EP30220-0008, and MOE-MOET32020-0004), NMRC Grant (NMRC/OFIRG/0076/2018), and Nanyang Assistant Professorship.

DNA topoisomerases modulate DNA topology to maintain chromosome superstructure and genome integrity, which is indispensable for DNA replication and RNA transcription. Their function in plant development still remains largely unknown. Here, we report a hitherto unidentified role of Topoisomerase Iα (TOP1α) in controlling flowering time in Arabidopsis (Arabidopsis thaliana). Loss of function of TOP1α results in early flowering under both long and short days. This is attributed mainly to a decrease in the expression of a central flowering repressor, FLOWERING LOCUS C (FLC), and its close homologs, MADS AFFECTING FLOWERING4 (MAF4) and MAF5, during the floral transition. TOP1α physically binds to the genomic regions of FLC, MAF4, and MAF5 and promotes the association of RNA polymerase II complexes to their transcriptional start sites. These correlate with the changes in histone modifications but do not directly affect nucleosome occupancy at these loci. Our results suggest that TOP1α mediates DNA topology to facilitate the recruitment of RNA polymerase II at FLC, MAF4, and MAF5 in conjunction with histone modifications, thus facilitating the expression of these key flowering repressors to prevent precocious flowering in Arabidopsis.

Cues of maternal and paternal origins interact to control seed development, and the underlying molecular mechanisms are still far from clear. Here, we show that TOPOISOMERASE Iα (TOP1α), UP-FRAMESHIFT SUPPRESSOR 1 (UPF1), and TRANSPARENT TESTA GLABRA2 (TTG2) gametophytically, biparentally regulate seed size in Arabidopsis. TOP1α and UPF1 are mainly expressed in antipodal cells, and loss of their function leads to ectopic TTG2 expression in these female gametophytic cells. We further demonstrate that TOP1α and UPF1 directly repress TTG2 expression through affecting its chromatin status and determine its relative expression in antipodal cells versus sperm cells, which controls seed size in a dosage-dependent and parent-of-origin-dependent manner. The molecular interplay among these three genes explains their biparental gametophytic effect during diploidy and interploidy reciprocal crosses. Taken together, our findings reveal a molecular framework of parental interaction for seed size control., Cues of maternal and paternal origin interact to control seed development, and the underlying molecular mechanisms are still far from clear. This study shows that in Arabidopsis, the relative dosage of the transcription factor TTG2 between antipodal cells and sperm cells at the beginning of seed development determines seed size under the control of TOP1α and UPF1.

Summary Plants exhibit different flowering behaviors in response to variable photoperiods across a wide geographical range. Here, we identify MYC3, a bHLH transcription factor, and its cis-element form the long-sought regulatory module responsible for cis-regulatory changes at the florigen gene FLOWERING LOCUS T (FT) that mediate natural variation in photoperiodic flowering responses in Arabidopsis. MYC3 is stabilized by DELLAs in the gibberellin pathway to suppress FT through binding the ACGGAT motif and antagonizing CONSTANS (CO) activation. Changing photoperiods modulate the relative abundance of MYC3 and CO, thus determining either of them as the predominant regulator for FT expression under different day lengths. Cis-regulatory changes in the MYC3 binding site at FT are associated with natural variation in day-length requirement for flowering in Arabidopsis accessions. Our findings reveal that environmental and developmental signals converge at MYC3 suppression of FT, an elementary event underlying natural variation in photoperiodic flowering responses.

DNA damage stabilizes and activates p53, which selectively induces downstream targets to modulate the cellular response. As a homeostatic regulator of cell cycle checkpoint, the p53 target Wip1 plays essential roles in releasing cells from DNA damage-induced checkpoints after appropriate repair of the damaged-DNA. It is unknown how Wip1 performs when the DNA damage is beyond repair. Here we address that Wip1 displays dose-dependent responses to UVC irradiation. A low dose of UVC, which stimulates intra-S phase cell cycle arrest, transiently induces the Wip1 protein levels in a p53-dependent manner. In contrast, a high dose of UVC, which induces apoptosis, suppresses the Wip1 protein levels in a p53-independent manner. The UVC dose-dependent response of Wip1 correlates not only with the cellular response but also with the activity of p53. Wip1 dephosphorylates p53 on its Ser15 residue. However, the mutual regulation between Wip1 and p53 is only triggered by a low dose of UVC. In response to a high dose of UVC, the sustained activation of p53 fails to induce the downstream targets, including Wip1, Mdm2, p21 and GADD45α. Nonetheless, the reduced Wip1 level contributes to the sustained accumulation of phospho-p53 (Ser15) in response to a high dose of UVC. Our results suggest that Wip1 is regulated by UVC in a dose-dependent manner. Moreover, the mutual regulation between Wip1 and p53 is highly dose-dependent upon UVC irradiation, and this contributes to the different outcomes of the cellular response to UVC.

We report that the Arabidopsis thaliana mutant sensitive to ABA and drought2 (sad2), which harbors a T-DNA insertion in an importin β-like gene, is more tolerant to UV-B radiation than the wild type. Analysis of cyclobutane pyrimidine dimer accumulation revealed that less DNA damage occurred in sad2 than in the wild type during UV-B treatment. No significant growth difference was observed between sad2 and the wild type when treated with the genotoxic drug methyl methanesulfonate, suggesting that SAD2 functions in UV-B protection rather than in DNA damage repair. Whereas the R2R3-type transcription repressor MYB4 has previously been shown to negatively regulate the transcription of cinnamate 4-hydroxylase (C4H) and thus to regulate the synthesis of sinapate esters, expression of both MYB4 and C4H and accumulation of UV-absorbing compounds were significantly higher in sad2 than in the wild type. MYB4 did not localize to the nucleus in the sad2 mutant, suggesting that SAD2 is required for MYB4 nuclear trafficking. SAD2 and MYB4 coimmunoprecipitated, indicating that these proteins localize in the same complex in vivo. MYB4 protein specifically bound to its own promoter in gel shift assays and repressed its own expression, demonstrating that MYB4 protein and mRNA are part of a negative autoregulatory loop. This feedback loop is altered in the sad2 mutant due to the absence of MYB4 protein in the nucleus, leading to the constitutive expression of MYB4 and C4H and resulting in accumulation of UV-absorbing pigments that shield the plant from UV-B radiation.

TOPOISOMERASE1 (TOP1), which releases DNA torsional stress generated during replication through its DNA relaxation activity, plays vital roles in animal and plant development. In Arabidopsis (Arabidopsis thaliana), TOP1 is encoded by two paralogous genes (TOP1α and TOP1β), of which TOP1α displays specific developmental functions that are critical for the maintenance of shoot and floral stem cells. Here, we show that maintenance of two different populations of root stem cells is also dependent on TOP1α-specific developmental functions, which are exerted through two distinct novel mechanisms. In the proximal root meristem, the DNA relaxation activity of TOP1α is critical to ensure genome integrity and survival of stele stem cells (SSCs). Loss of TOP1α function triggers DNA double-strand breaks in S-phase SSCs and results in their death, which can be partially reversed by the replenishment of SSCs mediated by ETHYLENE RESPONSE FACTOR115 In the quiescent center and root cap meristem, TOP1α is epistatic to RETINOBLASTOMA-RELATED (RBR) in the maintenance of undifferentiated state and the number of columella stem cells (CSCs). Loss of TOP1α function in either wild-type or RBR RNAi plants leads to differentiation of CSCs, whereas overexpression of TOP1α mimics and further enhances the effect of RBR reduction that increases the number of CSCs Taken together, these findings provide important mechanistic insights into understanding stem cell maintenance in plants.

Root gravitropism allows plants to establish root systems and its regulation depends on polar auxin transport mediated by PIN-FORMED (PIN) auxin transporters. PINOID (PID) and PROTEIN PHOSPHATASE 2A (PP2A) act antagonistically on reversible phosphorylation of PINs. This regulates polar PIN distribution and auxin transport. Here we show that a peptidyl-prolyl cis/trans isomerase Pin1At regulates root gravitropism. Downregulation of Pin1At suppresses root agravitropic phenotypes of pp2aa and 35S:PID, while overexpression of Pin1At affects root gravitropic responses and enhances the pp2aa agravitropic phenotype. Pin1At also affects auxin transport and polar localization of PIN1 in stele cells, which is mediated by PID and PP2A. Furthermore, Pin1At catalyses the conformational change of the phosphorylated Ser/Thr-Pro motifs of PIN1. Thus, Pin1At mediates the conformational dynamics of PIN1 and affects PID- and PP2A-mediated regulation of PIN1 polar localization, which correlates with the regulation of root gravitropism., Asymmetric transport of auxin allows plants to orient growth of their roots towards gravity. Here, the authors show that the peptidyl-prolyl cis/trans isomerase Pin1At contributes to root gravitropism and propose that it acts by altering the polarity of the PIN1 auxin transport protein.

The Lugol’s staining method has been widely used to detect changes in the maintenance of stem cell fate in the columella root cap of Arabidopsis roots since the late ‘90s. However, various limitations of this method demand for additional or complementary new approaches. For instance, it is unable to reveal the division rate of columella root cap stem cells. Here we report that, by labelling dividing stem cells with 5-ethynyl-2´-deoxyuridine (EdU), the number and distribution of their labeled progeny can be studied so that the division rate of stem cells can be measured quantitatively and in addition, that the progression of stem cell progeny differentiation can be assessed in combination with Lugol’s staining. EdU staining takes few hours and visualization of the stain characteristics of columella root cap can be performed easily under confocal microscopes. This simple technology, when used together with Lugol’s staining, provides a novel quantitative method to study the dynamics of stem cell behaviour that govern homeostasis in the Arabidopsis columella root cap.

Polycomb group proteins play essential roles in the epigenetic control of gene expression in plants and animals. Although some components of Polycomb repressive complex 1 (PRC1)-like complexes have recently been reported in the model plant Arabidopsis, how they contribute to gene repression remains largely unknown. Here we show that a putative PRC1 RING-finger protein, AtRING1A, plays a hitherto unknown role in mediating the transition from vegetative to reproductive development in Arabidopsis. Loss of function of AtRING1A results in the late-flowering phenotype, which is attributed to derepression of two floral repressors, MADS AFFECTING FLOWERING 4/5 (MAF4/5), which in turn downregulate two floral pathway integrators, FLOWERING LOCUS T and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1. Levels of the H3K27me3 repressive mark at MAF4 and MAF5 loci, which is deposited by CURLY LEAF (CLF)-containing PRC2-like complexes and bound by LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), are affected by AtRING1A, which interacts with both CLF and LHP1. Levels of the H3K4me3 activation mark correlate inversely with H3K27me3 levels at MAF4 and MAF5 loci. Our results suggest that AtRING1A suppresses the expression of MAF4 and MAF5 through affecting H3K27me3 levels at these loci to regulate the floral transition in Arabidopsis.

Most genes identified that control Arabidopsis trichome initiation and formation are transcription factors or regulatory components in transcriptional networks and include GLABROUS1 (GL1), GLABRA2 (GL2), GLABRA3 (GL3) and TRANSPARENT TESTA GLABRA1 (TTG1). Herein, we report that an importin beta-like protein, SENSITIVE TO ABA AND DROUGHT2 (SAD2), is required for trichome initiation. Mutations in SAD2 disrupted trichome initiation resulting in reduced trichome number, but had no effect on trichome development or root hair number and development. Expression levels of GL1, MYB23, GL2 and TTG1 were reduced in shoots of sad2 mutants while expression levels of GL3 and ENHANCER OF GLABRA3 (EGL3) were enhanced. Overexpression of GL3 increased trichome numbers in wild type but not in sad2 mutants, indicating that the function of the GL3 protein is altered in the sad2 mutants. In contrast, overexpression of GFP-GL1 decreased trichome number in both wild type and sad2. Double mutant analysis of gl1 sad2 and gl3 sad2 indicated that SAD2 functions genetically, at least in part, in the same pathway with these two genes. Co-immunoprecipitation indicated that the sad2 mutation does not disrupt formation of the TTG1-GL3-GL1 complex. Analysis of GFP fusions of GL1, GL2, GL3 and TTG1 suggested that these proteins are most likely not direct cargo of SAD2. Our data suggest that SAD2 is involved in trichome initiation by regulating these nuclear genes.

The genetic regulation of stomatal movement mainly depends on an efficient control system of gene expression, and guard cell-specific promoter is becoming the best choice. Here we combined the dehydration responsive element (DRE) with guard cell specific element (GCSE) to construct a novel promoter, DGP1. Histochemical assays in transgenic tobacco carrying beta, -glucuronidase (gus) gene fused to DGP1 demonstrated that GUS activity was found to be highly inducible by drought treatment and specifically restricted to guard cells. No GUS activity was detected in roots, stems or flowers after treatment. Further quantitative analysis showed that GUS activity in the epidermal strips was apparently induced by dehydration and dramatically increased with the elongation of treatment. The GUS activity after 8 h treatment was 179 times that of those without treatment. Although GUS activity in roots, stems or mesophyll increased after treatment, no great changes were observed. These results suggested that DGP1 could drive target gene expressed in guard cells when plant is subjected to drought stress. And this gets us prepared to control opening and closing of stomata through plant gene engineering.