Loss of function of OsDCL1 affects microRNA accumulation and causes developmental defects in rice

MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are two types of noncoding RNAs involved in developmental regulation, genome maintenance, and defense in eukaryotes. The activity of Dicer or Dicer-like (DCL) proteins is required for the maturation of miRNAs and siRNAs. In this study, we cloned and sequenced 66 candidate rice (Oryza sativa) miRNAs out of 1,650 small RNA sequences (19 to approximately 25 nt), and they could be further grouped into 21 families, 12 of which are newly identified and three of which, OsmiR528, OsmiR529, and OsmiR530, have been confirmed by northern blot. To study the function of rice DCL proteins (OsDCLs) in the biogenesis of miRNAs and siRNAs, we searched genome databases and identified four OsDCLs. An RNA interference approach was applied to knock down two OsDCLs, OsDCL1 and OsDCL4, respectively. Strong loss of function of OsDCL1IR transformants that expressed inverted repeats of OsDCL1 resulted in developmental arrest at the seedling stage, and weak loss of function of OsDCL1IR transformants caused pleiotropic developmental defects. Moreover, all miRNAs tested were greatly reduced in OsDCL1IR but not OsDCL4IR transformants, indicating that OsDCL1 plays a critical role in miRNA processing in rice. In contrast, the production of siRNA from transgenic inverted repeats and endogenous CentO regions were not affected in either OsDCL1IR or OsDCL4IR transformants, suggesting that the production of miRNAs and siRNAs is via distinct OsDCLs.     

Smad7 attenuates TGF-β-mediated aging-related hypofunction of submandibular glands

Submandibular glands have essential functions in taste, mastication, swallowing, and digestion. Submandibular gland hypofunction is prevalent in the elderly, impairing the patients’ quality of life. Current clinical treatment strategies have not decelerated or reversed the pathological process of submandibular gland hypofunction. Therefore, novel restoration strategies should be explored. However, studies on the mechanism of aging-related submandibular gland hypofunction remain very limited. The role of the TGF-β/Smad pathway in fibrosis has been studied in other organs. Therefore, this study aimed to elucidate the role of TGF-β/Smad signaling in the aging-related submandibular gland hypofunction. The results showed that Smad7 knockout in mice decreased the salivary flow rate. H&E, Masson trichrome, and immunohistochemistry staining of MCP-1 and α-SMA showed that Smad7 knockout in mice resulted in lymphocytic infiltration, acinar cell atrophy, and interstitial fibrosis. The Western blotting of collagen I and III also confirmed extensive fibrosis. We then found that Smad7 depletion resulted in the TGF-β-mediated fibrosis via mir-21, mir-29, and np_5318, and NFκB-driven inflammation activation. This study confirmed the inhibitory role of Smad7 in the aging-related submandibular gland hypofunction. Therefore, it provided a promising treatment target for aging-related dysfunction and sialadenitis of submandibular gland.     

Roles of DCL4 and DCL3b in rice phased small RNA biogenesis

Higher plants have evolved multiple proteins in the RNase III family to produce and regulate different classes of small RNAs with specialized molecular functions. In rice (Oryza sativa), numerous genomic clusters are targeted by one of two microRNAs (miRNAs), miR2118 and miR2275, to produce secondary small interfering RNAs (siRNAs) of either 21 or 24 nucleotides in a phased manner. The biogenesis requirements or the functions of the phased small RNAs are completely unknown. Here we examine the rice Dicer-Like (DCL) family, including OsDCL1, -3a, -3b and -4. By deep sequencing of small RNAs from different tissues of the wild type and osdcl4-1, we revealed that the processing of 21-nucleotide siRNAs, including trans-acting siRNAs (tasiRNA) and over 1000 phased small RNA loci, was largely dependent on OsDCL4. Surprisingly, the processing of 24-nucleotide phased small RNA requires the DCL3 homolog OsDCL3b rather than OsDCL3a, suggesting functional divergence within DCL3 family. RNA ligase-mediated 5' rapid amplification of cDNA ends and parallel analysis of RNA ends (PARE)/degradome analysis confirmed that most of the 21- and 24-nucleotide phased small RNA clusters were initiated from the target sites of miR2118 and miR2275, respectively. Furthermore, the accumulation of the two triggering miRNAs requires OsDCL1 activity. Finally, we show that phased small RNAs are preferentially produced in the male reproductive organs and are likely to be conserved in monocots. Our results revealed significant roles of OsDCL4, OsDCL3b and OsDCL1 in the 21- and 24-nucleotide phased small RNA biogenesis pathway in rice.     

Rice RNA-dependent RNA polymerase 6 acts in small RNA biogenesis and spikelet development

Higher plants have evolved multiple RNA-dependent RNA polymerases (RDRs), which work with Dicer-like (DCL) proteins to produce different classes of small RNAs with specialized molecular functions. Here we report that OsRDR6, the rice (Oryza sativa L.) homolog of Arabidopsis RDR6, acts in the biogenesis of various types and sizes of small RNAs. We isolated a rice osrdr6-1 mutant, which was temperature sensitive and showed spikelet defects. This mutant displays reduced accumulation of tasiR-ARFs, the conserved trans-acting siRNAs (tasiRNAs) derived from the TAS3 locus, and ectopic expression of tasiR-ARF target genes, the Auxin Response Factors (including ARF2 and ARF3/ETTIN). The loss of tasiR-mediated repression of ARFs in osrdr6-1 can explain its morphological defects, as expression of two non-targeted ARF3 gene constructs (ARF3muts) in a wild-type background mimics the osrdr6 and osdcl4-1 mutant phenotypes. Small RNA high-throughput sequencing also reveals that besides tasiRNAs, 21-nucleotide (nt) phased small RNAs are also largely dependent on OsRDR6. Unexpectedly, we found that osrdr6-1 has a strong impact on the accumulation of 24-nt phased small RNAs, but not on unphased ones. Our work uncovers the key roles of OsRDR6 in small RNA biogenesis and directly illustrates the crucial functions of tasiR-ARFs in rice development.     

CK2-Mediated Hyperphosphorylation of Topoisomerase I Targets Serine 506, Enhances Topoisomerase I–DNA Binding,and Increases Cellular Camptothecin Sensitivity

Topoisomerase I is the target for a potent class of chemotherapeutic drugs derived from the plant alkaloid camptothecin that includes irinotecan and topotecan. In this study we have identified a novel site of CK2-mediated topoisomerase I (topo I) phosphorylation at serine 506 (PS506) that is relevant to topo I function and to cellular responses to these topo I-targeted drugs. CK2 treatment induced hyperphosphorylation of recombinant topo I and expression of the PS506 epitope, and resulted in increased binding of topo I to supercoiled plasmid DNA. Hyperphosphorylated topo I was approximately three times more effective than the basal phosphorylated enzyme at relaxing plasmid supercoils but had similar DNA cleavage activity once bound to DNA. The PS506 epitope was expressed in cancer cell lines with elevated CK2 activity, hyperphosphorylated topo I, and increased sensitivity to camptothecin. In contrast, PS506 was not detected in normal cells or cancer cell lines with lower levels of CK2 activity. By experimentally manipulating CK2 activity in cancer cell lines, we demonstrate a cause and effect relationship between CK2 activity, PS506 expression, camptothecin-induced cellular DNA damage, and cellular camptothecin sensitivity. Our results show that the PS506 epitope is an indicator of dysregulated, hyperphosphorylated topo I in cancer cells, and may thus serve as a diagnostic or prognostic biomarker and predict tumor responsiveness to widely used topo I-targeted therapies.     

洪湖水体藻类藻相特征及其对生境的响应

藻类是水生态系统的重要成分,它的群落结构、细胞密度变化与水环境相适应,随水环境的变化而改变,因此藻相变化是评价水体质量的一项重要指标。2009—2010年在洪湖水面不同方位布点采样,对水体的浮游藻类藻相(群落结构、密度)和水质状况(水位(water level,H)、水温(water temperature,tw)、透明度(transparency,SD)、总氮(total nitrogen,TN)、总磷(totalphosphorus,TP)、高锰酸盐指数(permanganate index,CODMn)和叶绿素含量(chlorophyll a,Chl-a)等)进行了逐月的调查、监测,采用湖库营养状态指数法对湖泊富营养化评价,并运用多元逐步回归方法建立水体藻类与生境因子的回归方程。结果表明:洪湖水体总体上已达到轻度富营养化状态;藻类以蓝藻门、硅藻门和绿藻门为主,共鉴定出7门65属,藻细胞密度为1.14×106—3.24×107个/L,藻相季节变化特征明显。藻类组成以蓝藻门密度最高(52.93%),硅藻门(25.96%)和绿藻门(16.83%)次之,隐藻门(1.98%)、金藻门(1.64%)、裸藻门(0.42%)和甲藻门(0.25%)相对较低。藻类藻相及优势种在不同季节有所差异,冬春季节(12—5月)以硅藻门的直链藻、小环藻和针杆藻为优势种,夏秋季节(7—11月)以蓝藻门的鱼腥藻和微囊藻为优势种;湖泊藻类细胞密度与tw、CODMn呈正显著相关关系,与H、TN、TP、SD相关关系不显著;藻细胞生长逐步回归方程为y=3.7815+0.0794tw+0.5670CODMn-1.3467SD,藻细胞生长主要受水温和有机污染物等的影响,氮磷不是藻类生长的控制因子;在湖泊不同区域,环境条件存在差异,导致藻类生长过程中起主导作用的环境因子存在一定的差异,蓝藻生长主要受tw、CODMn和SD控制,绿藻生长主要受H、tw和CODMn影响,而硅藻生长决定于水体CODMn。     

Influence of polymerization temperature on the molecular recognition of imprinted polymers

This paper aimed at investigating the influence of polymerization temperature on the molecular recognition of molecularly imprinted polymers (MIPs) based on multiple non-covalent interactions. 3-l-Phenylalanylaminopyridine (3-l-PheNHPy) imprinted polymers were prepared using azobisnitriles as either thermal initiators or photoinitiators at various temperatures of 10, 40 and 60 degrees C, respectively. These polymers were subsequently evaluated in the high-performance liquid chromatographic (HPLC) mode for enantioselectivity. An unexpected result shows that polymer prepared at 40 degrees C has the highest enantioselectivity, but not the polymer prepared at lower temperature of 10 degrees C. Further, the effect of elution temperature and sample load on the selectivity of polymers was investigated in detail. In order to get a better understanding of the "exception", the influence of polymerization temperature on the polymerization extent and polymer morphology was studied by FT-IR spectrum test, cross-polarization magic angle spinning (CP-MAS) (13)NMR spectra experiment and pore analysis. Based on these results we attribute this "exception" to that there is a tradeoff between the extent of polymerization and stabilization of the template-functional monomer complexes. And an optimal polymerization temperature can be found for each combination of template and monomer.     

Global Identification of MicroRNAs and Their Targets in Barley under Salinity Stress

Salinity is a major limiting factor for agricultural production worldwide. A better understanding of the mechanisms of salinity stress response will aid efforts to improve plant salt tolerance. In this study, a combination of small RNA and mRNA degradome sequencing was used to identify salinity responsive-miRNAs and their targets in barley. A total of 152 miRNAs belonging to 126 families were identified, of which 44 were found to be salinity responsive with 30 up-regulated and 25 down-regulated respectively. The majority of the salinity-responsive miRNAs were up-regulated at the 8h time point, while down-regulated at the 3h and 27h time points. The targets of these miRNAs were further detected by degradome sequencing coupled with bioinformatics prediction. Finally, qRT-PCR was used to validate the identified miRNA and their targets. Our study systematically investigated the expression profile of miRNA and their targets in barley during salinity stress phase, which can contribute to understanding how miRNAs respond to salinity stress in barley and other cereal crops.     

Phytohormone Profiling During Tuber Development of Chinese Yam by Ultra-high performance Liquid Chromatography–Triple Quadrupole Tandem Mass Spectrometry

Changes in agronomic characters and the profile of various endogenous phytohormones during tuber development were studied in Dioscorea opposite (Chinese yam) cv. Guihuai 16. Tuber development exhibited a sigmoidal growth pattern according to the changes in tuber agronomic characters. The growth cycle of yam tuber could be divided into three stages: initiation stage, enlargement stage, and maturation stage. Moreover, the enlargement stage could be separated into three phases—slow growth phase, rapid growth phase, and late growth phase. Endogenous changes in phytohormones were associated with developmental changes in the tubers. The pulses of bioactive gibberellins (such as GA₃ and GA₄) were measured in tubers. The highest contents of GA₃ and GA₄ were reached 90 days after field planting, corresponding to the beginning of the rapid growth phase of tuber enlargement. Changes in trans-zeatin (tZ), jasmonic acid (JA), indole-3-acetic acid (IAA), and abscisic acid (ABA) levels were also observed, and seemed to be related to tuber enlargement at different phases. Continuous increases in JA and tZ contents accompanied tuber enlargement. Transient pulses of both IAA and ABA contents were also observed at the start of tuber rapid growth. Additionally, a second peak level of IAA was detected at the tuber maturation stage. These results suggest GAs play a key role at the beginning of the tuber rapid growth stage, and there is a close relationship between whole tuber enlargement and the contents of JA and tZ. Moreover, it is suggested that IAA and ABA also may be linked to the beginning of tuber rapid growth, and IAA also seems to be correlated to late tuber maturation.