miR824-Regulated AGAMOUS-LIKE16 Contributes to Flowering Time Repression in Arabidopsis

The timing of flowering is pivotal for maximizing reproductive success under fluctuating environmental conditions. Flowering time is tightly controlled by complex genetic networks that integrate endogenous and exogenous cues, such as light, temperature, photoperiod, and hormones. Here, we show that AGAMOUS-LIKE16 (AGL16) and its negative regulator microRNA824 (miR824) control flowering time in Arabidopsis thaliana. Knockout of AGL16 effectively accelerates flowering in nonvernalized Col-FRI, in which the floral inhibitor FLOWERING LOCUS C (FLC) is strongly expressed, but shows no effect if plants are vernalized or grown in short days. Alteration of AGL16 expression levels by manipulating miR824 abundance influences the timing of flowering quantitatively, depending on the expression level and number of functional FLC alleles. The effect of AGL16 is fully dependent on the presence of FLOWERING LOCUS T (FT). Further experiments show that AGL16 can interact directly with SHORT VEGETATIVE PHASE and indirectly with FLC, two proteins that form a complex to repress expression of FT. Our data reveal that miR824 and AGL16 modulate the extent of flowering time repression in a long-day photoperiod.     

Pre-miR172及miR172调控油菜AP2基因表达的规律分析

为探究油菜miR172前体(pre-miR172)及成熟体(miR172)对AP2基因的调控功能,该研究通过生物信息学方法对miR172和AP2启动子进行调控元件预测,分析6条油菜AP2基因的进化关系及miR172与AP2的靶向关系; 通过qRT-PCR方法检测AP2、miR172和pre-miR172在早熟和晚熟油菜不同组织的表达规律; 比较分析miR172丰度和AP2表达量间的相关关系,以及比较分析 pre-miR172和miR172在表达水平上的相关关系; 通过过表达pre-miR172,再次验证pre-miR172对成熟体miR172及AP2的作用。结果表明:(1)miR172和AP2启动子区均存在调控花发育的顺式元件。(2)6条AP2序列均经历了强烈的纯化选择,均具备miR172的结合位点,属miR172的靶基因。(3)miR172家族成员均可促进早熟油菜AP2表达,但miR172d作用不明显。在晚熟油菜中,miR172a和miR172c作用微弱,miR172b和miR172d二者共同发挥作用降低AP2的表达水平。(4)pre-miR172家族对于早熟油菜中miR172家族的表达水平均有促进作用; 在晚熟油菜中pre-miR172a和pre-miR172b对其成熟序列的形成发挥正调控作用,pre-miR172c和pre-miR172d则对于其成熟序列的形成发挥负调控作用。过表达pre-miR172后,miR172和AP2表达规律与上述结果保持一致,证实pre-miR172对miR172及AP2的调控功能。该研究结果丰富了油菜AP2基因的功能调控路径,为基因的调控功能研究提供了新的思路。     

miR172: a messenger between nodulation and flowering

Legumes coordinate nodulation and plant development to maximize reproductive success, but the underlying molecular mechanisms are not well understood. A recent study by Yun et al. has revealed that nodulation drives root-to-shoot movement of microRNA172 (miR172) to accelerate flowering time, thus building a new bridge between nodulation and plant growth regulation.     

Recovery of dicer-like 1-late flowering phenotype by miR172 expressed by the noncanonical DCL4-dependent biogenesis pathway

MicroRNAs (miRNAs) act as down-regulators of gene expression, and play a dominant role in eukaryote development. In Arabidopsis thaliana, DICER-LIKE 1 (DCL1) is the main processor in miRNA biogenesis, and dcl1 mutants show various developmental defects at the early stage of embryogenesis or at gamete formation. However, miRNAs responsible for the respective developmental stages of the dcl1 defects have not been identified. Here, we developed a DCL1-independent miRNA expression system using the unique DCL4-dependent miRNA, miR839. By replacing the mature sequence in the miR839 precursor sequence with that of miR172, one of the most widely conserved miRNAs in angiosperms, we succeeded in expressing miR172 from a chimeric miR839 precursor in dcl1-7 plants and observed the repression of miR172 target gene expression. In parallel, the DCL4-dependent miR172 expression rescued the late flowering phenotype of dcl1-7 by acceleration of flowering. We established the DCL1-independent miRNA expression system, and revealed that the reduction of miR172 expression is responsible for the dcl1-7 late flowering phenotype.     

PTLF-PTAG-IR对转基因烟草开花的抑制效应

PTLF(LEAFY同源基因)和PTAG(AGAMOUS同源基因)是杨树中控制花发育的重要基因,为了解RNA干涉同时抑制PTLF和PTAG对花发育的影响,本研究采用根癌农杆菌(Agrobacterium tumefaciens)介导法将35S::PTLF-PTAG-IR导入烟草(Nicotiana tabacum)。经PCR检测获得了15株阳性转化株系,进一步的Southern分析证实PTLF-PTAG-IR基因已整合到烟草基因组中。荧光定量分析结果显示,转基因烟草内源NFL(LEAFY同源基因)和NAG1(AGAMOUS同源基因)的表达量均低于野生型。对开花时间调查的结果表明:与野生型烟草相比,53.3%的转基因株系开花受到完全的抑制,26.7%的转基因株系开花时间平均推迟34.3d,仅20%的转基因株系与野生型在同一时期开花。另外,对转基因烟草花器官的观察发现花瓣形态,雄蕊着生方式、数目等方面发生变异,这些研究结果表明过量表达35S::PTLF-PTAG-IR对烟草花发育起到抑制作用,本研究将为利用转基因手段获得不育材料提供参考。     

芥蓝miR172家族成员进化特性比较及时空表达分析

为了明确植物miR172家族成员的进化特性及其在不同组织部位的表达情况,该研究以芥蓝miR172a家族为例,通过芥蓝miR172a成熟体序列比对、前体(pre-miRNA)二级结构预测、系统发育进化树构建、靶基因预测及实时荧光定量等手段,对芥蓝miR172a和miR172b基因家族的进化特性及其在芥蓝不同组织部位中的表达规律进行分析。结果显示:(1)芥蓝miR172家族存在20个成熟体成员,通过比对发现20条序列都存在一个重叠区域,该区域中ACTAGATC 8个碱基高度保守,并且在芥蓝pre-miR172的3′和5′端均能形成成熟体。(2)mfold预测结果显示,miR172a家族5个前体成员的最小折叠自由能在-54.55~-78.60kal/mol之间,均能自发形成典型、稳定的茎环二级结构。(3)系统发育进化树显示,芥蓝miR172a家族的前体成员具有一定的保守性和多样性,并与番木瓜pre-miR172a亲缘关系较近。(4)靶基因预测发现芥蓝miR172a共有13个不同靶标基因,多个成员也可以作用于相同靶标基因。(5)实时荧光定量PCR显示,芥蓝pre-miR172a和pre-miR172b家族不同成员在不同组织部位表达量差异显著,10个成员在9个组织部位中的表达各不相同。其中,芥蓝pre-miR172a-1、pre-miR172a-2、premiR172a-3和pre-miR172b在芥蓝荚中大量表达;pre-miR172b-5p和pre-miR172b-5p-2在芥蓝花中大量表达。研究表明,miR172在芥蓝花和荚的发育过程中起着重要的作用。     

miR172-AP2模块调控植物生长发育及逆境响应的研究进展

MicroRNA (miRNA)是一类具有调控能力的非编码小分子RNA, 通过与靶基因mRNA特异或非特异性结合, 诱导靶基因mRNA降解或抑制其翻译, 从而调控植物的生长发育。其中, miR172的靶基因AP2所编码的转录因子为植物所特有, miR172在转录后或翻译水平对AP2进行表达调控, 进而调控植物的花发育、时序转换、小穗形态、块茎和果实发育、结瘤(豆科)以及逆境响应等过程。该文综述了近年来miR172-AP2模块在植物生长发育调控方面的最新研究进展。     

Reduced Soybean Water Stress Tolerance by miR393a-Mediated Repression of GmTIR1 and Abscisic Acid Accumulation

MicroRNA393 (miR393) has been shown to regulate plant water stress tolerance through an auxin signaling pathway. However, its role in soybean (Glycine max [L.] Merr.) has not yet been reported. Here, we examined the expression pattern of miR393 family members and their target gene GmTIR1 in water-stressed roots. Subsequently, we analyzed the functions of miR393 in the regulation of water stress tolerance and its relationship with GmTIR1 and abscisic acid (ABA) using a transgenic hairy root assay. Under water stress, miR393 family genes exhibited diverse expression patterns. Overexpression and knockdown analysis demonstrated that miR393a reduced water stress tolerance as measured by root vigor, net photosynthetic rate (Pn), and relative water content (RWC). Moreover, miR393a also caused down-regulation of GmTIR1A and GmTIR1B expression, an early decrease in hydrogen peroxide (H₂O₂) levels, early and late declines in ABA content and antioxidant activities, and a late elevation of H₂O₂ and malondialdehyde (MDA) concentrations in stressed hairy roots. However, overexpression and RNAi analyses showed that GmTIR1A and GmTIR1B triggered an early increase in H₂O₂, a rise in antioxidant activities during the early and late stages, a late decline in H₂O₂ and MDA contents, and a rise in root vigor, Pn, and RWC under water stress. Similarly, exogenously supplied ABA caused early H₂O₂ accumulation, early and late increases in antioxidant capacity, and a late decrease in oxidative damage in stressed miR393a-overexpressing roots. Therefore, our study presents a valuable model in which miR393a prevents early GmTIR1- and ABA-dependent increases in H₂O₂ and thus triggers a rise in antioxidant capacity, root vigor, RWC, and Pn, consequently decreasing water stress tolerance.

     

miR172 signals are incorporated into the miR156 signaling pathway at the SPL3/4/5 genes in Arabidopsis developmental transitions

In plants, developmental timing is coordinately regulated by a complex signaling network that integrates diverse intrinsic and extrinsic signals. miR172 promotes photoperiodic flowering. It also regulates adult development along with miR156, although the molecular mechanisms underlying this regulation are not fully understood. Here, we demonstrate that miR172 modulates the developmental transitions by regulating the expression of a subset of the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes, which are also regulated by miR156. The SPL3/4/5 genes were upregulated in the miR172-overproducing plants (35S:172) and its target gene mutants that exhibit early flowering. In contrast, expression of other SPL genes was not altered to a discernible level. Kinetic measurements of miR172 abundance in the transgenic plants expressing the MIR156a gene driven by a β-estradiol-inducible promoter revealed that expressions of miR172 and miR156 are not directly interrelated. Instead, the 2 miRNA signals are integrated at the SPL3/4/5 genes. Notably, analysis of developmental patterns in the 156?×?172 plants overproducing both miR172 and miR156 showed that whereas vegetative phase change was delayed as observed in the miR156-overproducing plants (35S:156), flowering initiation was accelerated as observed in the 35S:172 transgenic plants. Together, these observations indicate that although miR172 and miR156 play distinct roles in the timing of developmental phase transitions, there is a signaling crosstalk mediated by the SPL3/4/5 genes.     

Overexpression of miR172 suppresses the brassinosteroid signaling defects of bak1 in Arabidopsis

BRI1-Associated Receptor Kinase 1 (BAK1) is a leucine-rich repeat serine/threonine receptor-like kinase (LRR-RLK) that is involved in multiple developmental pathways, such as brassinosteroid (BR) signaling, plant immunity and cell death control in plants. Because the roundish and compact rosette leaves of bak1 mutant plants are characteristic phenotypes for deficient BR signaling, we screened genetic suppressors of bak1 according to changes in leaf shape to identify new components that may be involved in BAK1-mediated BR signaling using the activation-tagging method. Here, we report bak1-SUP1, which exhibited longer and narrower rosette leaves and an increased BR sensitivity compared with those of bak1. Analyses of the T-DNA insertional site and the gene expression that was affected by the T-DNA insertion revealed that a microRNA, namely, miR172, over-accumulates in bak1-SUP1. Detailed phenotypic analyses of bak1-SUP1 and a single mutant in which the bak1 mutation was segregated out (miR172-D) revealed that the overexpression of miR172 promotes leaf length elongation in adult plants and increases the root and hypocotyl growth during the seedling stage compared with that of wild type plants. Taken together with its increased BR sensitivity, these results suggest that miR172 regulates vegetative growth patterns by modulating BR sensitivity as well as by the previously identified developmental phase transition.     

Target Repression Induced by Endogenous microRNAs: Large Differences,Small Effects

MicroRNAs are small RNAs that regulate protein levels. It is commonly assumed that the expression level of a microRNA is directly correlated with its repressive activity – that is, highly expressed microRNAs will repress their target mRNAs more. Here we investigate the quantitative relationship between endogenous microRNA expression and repression for 32 mature microRNAs in Drosophila melanogaster S2 cells. In general, we find that more abundant microRNAs repress their targets to a greater degree. However, the relationship between expression and repression is nonlinear, such that a 10-fold greater microRNA concentration produces only a 10% increase in target repression. The expression/repression relationship is the same for both dominant guide microRNAs and minor mature products (so-called passenger strands/microRNA* sequences). However, we find examples of microRNAs whose cellular concentrations differ by several orders of magnitude, yet induce similar repression of target mRNAs. Likewise, microRNAs with similar expression can have very different repressive abilities. We show that the association of microRNAs with Argonaute proteins does not explain this variation in repression. The observed relationship is consistent with the limiting step in target repression being the association of the microRNA/RISC complex with the target site. These findings argue that modest changes in cellular microRNA concentration will have minor effects on repression of targets.     

Upregulation of miR21 and Repression of Grhl3 by Leptin Mediates Sinusoidal Endothelial Injury in Experimental Nonalcoholic Steatohepatitis

Sinusoidal endothelial dysfunction (SED) has been found to be an early event in nonalcoholic steatohepatitis (NASH) progression but the molecular mechanisms underlying its causation remains elusive. We hypothesized that adipokine leptin worsens sinusoidal injury by decreasing functionally active nitric oxide synthase 3 (NOS)3 via miR21. Using rodent models of NASH, and transgenic mice lacking leptin and leptin receptor, results showed that hyperleptinemia caused a 4–5 fold upregulation of hepatic miR21 as assessed by qRTPCR. The upregulation of miR21 led to a time-dependent repression of its target protein Grhl3 levels as shown by western blot analyses. NOS3-p/NOS3 ratio which is controlled by Grhl3 was significantly decreased in NASH models. SED markers ICAM-1, VEGFR-2, and E-selectin as assessed by immunofluorescence microscopy were significantly up regulated in the progressive phases of NASH. Lack of leptin or its receptor in vivo, reversed the upregulation of miR21 and restored the levels of Grhl3 and NOS3-p/NOS3 ratio coupled with decreased SED dysfunction markers. Interestingly, leptin supplementation in mice lacking leptin, significantly enhanced miR21 levels, decreased Grhl3 repression and NOS3 phosphorylation. Leptin supplementation in isolated primary endothelial cells, Kupffer cells and stellate cells showed increased mir21 expression in stellate cells while sinusoidal injury was significantly higher in all cell types. Finally miR21 KO mice showed increased NOS3-p/NOS3 ratio and reversed SED markers in the rodent models of NASH. The experimental results described here show a close association of leptin-induced miR21 in aiding sinusoidal injury in NASH.