A transcriptional repressor HVA regulates vascular bundle formation through auxin transport in Arabidopsis stem.

Vascular bundles transport water and photosynthate to all organs, and increased bundle number contributes to crop lodging resistance. However, the regulation of vascular bundle formation is poorly understood in the Arabidopsis stem. We report a novel semi-dominant mutant with high vascular activity, hva-d, showing increased vascular bundle number and enhanced cambium proliferation in the stem. The activation of a C2H2 zinc finger transcription factor, AT5G27880/HVA, is responsible for the hva-d phenotype. Genetic, biochemical, and fluorescent microscopic analyses were used to dissect the functions of HVA. HVA functions as a repressor and interacts with TOPLESS via the conserved Ethylene-responsive element binding factor-associated Amphiphilic Repression motif. In contrast to the HVA activation line, knockout of HVA function with a CRISPR-Cas9 approach or expression of HVA fused with an activation domain VP16 (HVA-VP16) resulted in fewer vascular bundles. Further, HVA directly regulates the expression of the auxin transport efflux facilitator PIN1, as a result affecting auxin accumulation. Genetics analysis demonstrated that PIN1 is epistatic to HVA in controlling bundle number. This research identifies HVA as a positive regulator of vascular initiation through negatively modulating auxin transport and sheds new light on the mechanism of bundle formation in the stem.

Effect of Comprehensive Care Based on Appropriate Chinese Medicine Techniques on Urinary Retention and Bladder Function Recovery after Total Hysterectomy in Patients with Cervical Cancer.

Objective: To investigate the effect of comprehensive care based on appropriate Chinese medicine techniques on urinary retention and bladder function recovery after total hysterectomy in patients with cervical cancer. Methods: A total of 148 cases admitted after radical hysterectomy for cervical cancer from January 2019 to early September 2019 were used as the observation sample and were divided into control and experimental groups based on a randomized double-blind method. There were 74 cases each. The control group was given comprehensive care, and the experimental group was given comprehensive care based on appropriate Chinese medicine techniques. The intervention period was 2 weeks after surgery. The recovery rate of bladder function and the occurrence of urinary retention were compared between the two groups, and the duration of postoperative retention of urinary catheter, the amount of residual urine, and the feeling of urination were counted. Results: The experimental group had better urinary catheter retention time, time to first spontaneous voiding, time to get out of bed, and time to anal discharge than the control group; the experimental group had a higher rate of good bladder function recovery than the control group and better bladder recovery time, residual urine volume, and incidence of urinary retention than the control group; the patients in the experimental group had better UDI-6 scores. Conclusion: The implementation of comprehensive care based on appropriate Chinese medicine techniques can relieve patients' difficulty in urination and improve their quality of life.

The XVP/ NAC003 protein associates with the plasma membrane through KR rich regions and translocates to the nucleus by changing phosphorylation status.

Membrane localized transcription factors play essential roles in various plant developmental processes. The XVP/NAC003 protein is a NAC domain transcription factor associated with the plasma membrane and involved in the TDIF-PXY signaling during vascular development. We report here the mechanisms of XVP membrane localization and its nuclear translocation. Using a transient transformation approach, we found that XVP is associated with the plasma membrane through positively charged KR-rich regions. Mutagenesis studies found that the threonine amino acid at position 354 (T354) is critical for XVP translocation to the nucleus. In particular, the threonine to alanine mutation (T354A) resulted in a partial nucleus localization, while threonine to aspartic acid (T354D) mutation showed no effect on protein localization, indicating that dephosphorylation at T354 may serve as a nucleus translocation signal. This research sheds new light on the nucleus partitioning of plasma membrane-associated transcription factors.

Identification and Characterization of a Pear Chlorotic Leaf Spot-Associated Virus, a Novel Emaravirus Associated with a Severe Disease of Pear Trees in China.

Pear chlorotic leaf spot (PCLS) is a recently emerged disease of commercially cultivated sandy pear (Pyrus pyrifolia) trees in central and southern China. By integrating high-throughput sequencing and conventional Sanger sequencing of reverse-transcription (RT)-PCR products, a novel emaravirus infecting pear trees was identified and molecularly characterized. The virus was provisionally named pear chlorotic leaf spot-associated virus (PCLSaV). PCLSaV shows the typical molecular features of members of the genus Emaravirus in the family Fimoviridae. It has a genome composed of at least five negative-sense RNA segments, with each containing a single open reading frame and two complementary 13-nucleotide stretches at the 5' and 3' termini. PCLSaV shows a close phylogenetic relationship with recognized emaraviruses but forms a separate clade. Moreover, double-membrane-bound bodies were observed in PCLSaV-infected tissues and in extracts of PCLSaV-infected leaves. For the first time, our study revealed the profile distribution of viral RNA reads from the RNA-seq libraries of three samples along the RNA1 to RNA5 of an emaravirus. Field surveys combined with specific RT-PCR assays revealed the presence of PCLSaV in almost all PCLS-diseased pear samples, strongly supporting the association of the virus with the PCLS disease. This study revealed the first emaravirus infecting pear trees and its association with a severe pear chlorotic leaf disease.

Activation of ACS7 in Arabidopsis affects vascular development and demonstrates a link between ethylene synthesis and cambial activity.

Ethylene is a gaseous hormone that affects many processes of plant growth and development. During vascular development, ethylene positively regulates cambial cell division in parallel with tracheary element differentiation inhibitory factor (TDIF) peptide signaling. In this study, we identified an ethylene overproducing mutant, acs7-d, exhibiting enhanced cambial activity and reduced wall development in fiber cells. Using genetic analysis, we found that ethylene signaling is necessary for the phenotypes of enhanced cambial cell division as well as defects in stem elongation and fiber cell wall development. Further, the cambial cell proliferation phenotype of acs7-d depends on WOX4, indicating that the two parallel pathways, ethylene and TDIF signaling, converge at WOX4 in regulating cambium activity. Gene expression analysis showed that ethylene impedes fiber cell wall biosynthesis through a conserved hierarchical transcriptional regulation. These results advance our understanding of the molecular mechanisms of ethylene in regulating vascular meristem activity.

Characterization of the Auxin Efflux Transporter PIN Proteins in Pear.

PIN-FORMED (PIN) encodes a key auxin polar transport family that plays a crucial role in the outward transport of auxin and several growth and development processes, including dwarfing trees. We identified a dwarfing pear rootstock 'OHF51' (Pyrus communis), which limits the growth vigor of the 'Xueqing' (Pyrus bretschneideri × Pyrus pyrifolia) scion, and isolated 14 putative PbPINs from the pear Pyrus bretschneideri. The phylogenic relationships, structure, promoter regions, and expression patterns were analyzed. PbPINs were classified into two main groups based on the protein domain structure and categorized into three major groups using the neighbor-joining algorithm. Promoter analysis demonstrated that PbPINs might be closely related to plant growth and development. Through quantitative real-time PCR (qRT-PCR) analysis, we found that the expression patterns of 14 PbPINs varied upon exposure to different organs in dwarfing and vigorous stocks, 'OHF51' and 'QN101' (Pyrus betulifolia), indicating that they might play varying roles in different tissues and participated in the regulation of growth vigor. These results provide fundamental insights into the characteristics and evolution of the PINs family, as well as the possible relationship between dwarfing ability and auxin polar transport.

Heavy metal copper accelerates the conjugative transfer of antibiotic resistance genes in freshwater microcosms.

Recent studies have consistently demonstrated increasing abundances of antibiotic resistance genes (ARGs) in the absence of antibiotic use. There is a large amount of quantitative data that has correlated the elevated ARGs levels with the concentrations of heavy metals in environments with anthropogenic impact. However, the mechanisms by which heavy metals facilitate the proliferation and horizontal gene transfer of ARGs among environmental bacteria were still unknown. This study validated effects of four typical heavy metals (Cu, Cd, Pb, Zn) on the plasmid RP4 mediated conjugative transfer of ARGs in freshwater microcosms. The results suggested that the typical heavy metals including Cu, Pb and Zn would promote conjugative transfer of the plasmid RP4, and Cu (5.0 µg/L) had the greatest ability to increase conjugative transfer by 16-fold higher than the control groups. In conjugative transfer microcosms, the species of each cultivable transconjugant were isolated, and their minimum inhibitory concentrations (MICs) were assessed via antibiotic susceptibility testing. The mechanism of the increased conjugative transfer of Cu was that Cu induced cell damage and the reduced conjugative transfer of Cd was that Cd increased the content of extracellular polymers substances (EPS). This study confirms that heavy metal Cu facilitates the conjugative transfer of environmental-mediated plasmid RP4 by cell damage effect, therefore accelerating the transmission and proliferation of ARGs.

A membrane-associated NAC domain transcription factor XVP interacts with TDIF co-receptor and regulates vascular meristem activity.

Vascular stem cell maintenance is regulated by a peptide signaling involving Tracheary Element Differentiation Inhibitory Factor (TDIF) and Receptor TDR/PXY (Phloem intercalated with Xylem) and co-receptor BAK1 (BRI1-associated receptor kinase1). The regulatory mechanism of this signaling pathway is largely unknown despite its importance in stem cell maintenance in the vascular meristem. We report that activation of a NAC domain transcription factor XVP leads to precocious Xylem differentiation, disruption of Vascular Patterning, and reduced cell numbers in vascular bundles. We combined molecular and genetic studies to elucidate the biological functions of XVP. XVP is expressed in the cambium, localized on the plasma membrane and forms a complex with TDIF co-receptors PXY-BAK1. Simultaneous mutation of XVP and its close homologous NAC048 enhances TDIF signaling. In addition, genetics analysis indicated that XVP promotes xylem differentiation through a known master regulator VASCULAR-RELATED NAC-DOMAIN6 (VND6). Expression analyses indicate that XVP activates CLAVATA3/ESR (CLE)-related protein 44 (CLE44), the coding gene of TDIF, whereas TDIF represses XVP expression, suggesting a feedback mechanism. Therefore, XVP functions as a negative regulator of the TDIF-PXY module and fine-tunes TDIF signaling in vascular development. These results shed new light on the mechanism of vascular stem cell maintenance.

LBD29-Involved Auxin Signaling Represses NAC Master Regulators and Fiber Wall Biosynthesis.

NAM, ATAF1/2 and CUC2 (NAC) domain transcription factors function as master switches in regulating secondary cell wall (SCW) biosynthesis in Arabidopsis (Arabidopsis thaliana) stems. Despite the importance of these NACs in fiber development, the upstream signal is still elusive. Using a large-scale mutant screening, we identified a dominant activation-tagging mutant, fiberless-d (fls-d), showing defective SCW development in stem fibers, similar to that of the nac secondary wall thickening promoting factor1-1 (nst1-1)nst3-3 double mutant. Overexpression of LATERAL ORGAN BOUNDARIES DOMAIN29 (LBD29) is responsible for the fls-d mutant phenotypes. By contrast, loss-of-function of LBD29, either in the dominant repression transgenic lines or in the transfer-DNA (T-DNA) insertion mutant lbd29-1, enhanced SCW development in fibers. Genetic analysis and transgenic studies demonstrated LBD29 depends on master regulators in mediating SCW biosynthesis, specifically NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 (NST1), NST2, and NST3. Increasing indole-3-acetic acid (IAA) levels, either in stem tissues above a N-1-naphthylphthalamic acid-treated region or in plants directly sprayed with IAA, inhibits fiber wall thickening. The inhibition effect of naphthylphthalamic acid treatment and exogenous IAA application depends on a known auxin signaling pathway involving AUXIN RESPONSE FACTOR7 (ARF7)/ARF19 and LBD29. These results demonstrate auxin is upstream of LBD29 in repressing NAC master regulators, and therefore shed new light on the regulation of SCW biosynthesis in Arabidopsis.

The α-Aurora Kinases Function in Vascular Development in Arabidopsis.

The Aurora kinases are serine/threonine kinases with conserved functions in mitotic cell division in eukaryotes. In Arabidopsis, Aurora kinases play important roles in primary meristem maintenance, but their functions in vascular development are still elusive. We report a dominant xdi-d mutant showing the xylem development inhibition (XDI) phenotype. Gene identification and transgenic overexpression experiments indicated that the activation of the Arabidopsis Aurora 2 (AtAUR2) gene is responsible for the XDI phenotype. In contrast, the aur1-2 aur2-2 double mutant plants showed enhanced differentiation of phloem and xylem cells, indicating that the Aurora kinases negatively affect xylem differentiation. The transcript levels of key regulatory genes in vascular cell differentiation, i.e. ALTERED PHLOEM DEVELOPMENT (APL), VASCULAR-RELATED NAC-DOMAIN 6 (VND6) and VND7, were higher in the aur1-2 aur2-2 double mutant and lower in xdi-d mutants compared with the wild-type plants, further supporting the functions of α-Aurora kinases in vascular development. Gene mutagenesis and transgenic studies showed that protein phosphorylation and substrate binding, but not protein dimerization and ubiquitination, are critical for the biological function of AtAUR2. These results indicate that α-Aurora kinases play key roles in vascular cell differentiation in Arabidopsis.

Activation of miR165b represses AtHB15 expression and induces pith secondary wall development in Arabidopsis.

Secondary cell-wall thickening takes place in sclerenchyma cells, but not in surrounding parenchyma cells. The molecular mechanism of switching on and off secondary wall synthesis in various cell types is still elusive. Here, we report the identification of a dominant mutant stp-2d showing secondary wall thickening in pith cells (STP). Immunohistochemistry assays confirmed accumulation of secondary cell walls in the pith cells of the stp-2d mutant. Activation of microRNA 165b (miR165b) expression is responsible for the STP phenotype, as demonstrated by transgenic over-expression experiments. The expression of three class III HD-ZIP transcription factor genes, including AtHB15, was repressed in the stp-2d mutant. Transgenic over-expression of a mutant form of AtHB15 that is resistant to miR165-mediated cleavage reversed the stp-2d mutant phenotype to wild-type, indicating that AtHB15 represses secondary wall development in pith. Characterization of two athb15 mutant alleles further confirmed that functional AtHB15 is necessary for retaining primary walls in parenchyma pith cells. Expression analyses of cell-wall synthetic genes and wall-related transcription factors indicated that a transcriptional pathway is involved in AtHB15 function. These results provide insight into the molecular mechanism of secondary cell-wall development.

Developmental analysis of a Medicago truncatula smooth leaf margin1 mutant reveals context-dependent effects on compound leaf development.

Compound leaf development requires highly regulated cell proliferation, differentiation, and expansion patterns. We identified loss-of-function alleles at the SMOOTH LEAF MARGIN1 (SLM1) locus in Medicago truncatula, a model legume species with trifoliate adult leaves. SLM1 encodes an auxin efflux carrier protein and is the ortholog of Arabidopsis thaliana PIN-FORMED1 (PIN1). Auxin distribution is impaired in the slm1 mutant, resulting in pleiotropic phenotypes in different organs. The most striking change in slm1 is the increase in the number of terminal leaflets and a simultaneous reduction in the number of lateral leaflets, accompanied by reduced expression of SINGLE LEAFLET1 (SGL1), an ortholog of LEAFY. Characterization of the mutant indicates that distinct developmental domains exist in the formation of terminal and lateral leaflets. In contrast with the pinnate compound leaves in the wild type, the slm1 sgl1 double mutant shows nonpeltately palmate leaves, suggesting that the terminal leaflet primordium in M. truncatula has a unique developmental mechanism. Further investigations on the development of leaf serrations reveal different ontogenies between distal serration and marginal serration formation as well as between serration and leaflet formation. These data suggest that regulation of the elaboration of compound leaves and serrations is context dependent and tightly correlated with the auxin/SLM1 module in M. truncatula.