'Evidence of an auxin signal pathway, microRNA167-ARF8-GH3, and its response to exogenous auxin in cultured rice cells'

MicroRNA167 (miR167) was shown to cleave auxin responsive factor 8 (ARF8) mRNA in cultured rice cells. MiR167 level was found to be controlled by the presence of auxin in the growth medium. When cells grew in auxin-free medium, miR167 level decreased, resulting in an increase in the level of ARF8 mRNA. Cells growing in the normal growth medium containing auxin showed a reversed trend. It was also shown that expression of OsGH3-2, an rice IAA-conjugating enzyme, was positively regulated by ARF8. Delivery of synthesized miR167 into cells led to decrease of both ARF8 mRNA and OsGH3-2 mRNA. This study provides an evidence in which the exogeneous auxin signal is transduced to OsGH3-2 through miR167 and ARF8 in sequence. This proposed auxin signal transduction pathway, auxin-miR167-ARF8-OsGH3-2, could be, in conjunction with the other microRNA-mediated auxin signals, an important one for responding to exogeneous auxin and for determining the cellular free auxin level which guides appropriate auxin responses.     

A set of Arabidopsis thaliana miRNAs involve shoot regeneration in vitro

Plant miRNAs, the critical regulator of gene expression, involve many development processes in vivo. However, the roles of miRNAs in plant cell proliferation and redifferntiation in vitro remain unknown. To determine better the molecular mechanism of these processes, we have recently reported that a set of miRNAs with different expression patterns between cells of totipotent and non-totipotent Arabidopsis calli. Some of these were specifically up- or downregulated during callus formation or shoot regeneration, and other development. Among them, miR160, and one of its target genes, ARF10, regulated Arabidopsis in vitro shoot regeneration via WUS, CLV3 and CUC1/2. The miR160-overexpressing, 35S transgenic lines, exhibited reduced shoot regeneration efficiency. The mARF10, a miR160-resistant form of ARF10, showed a high level of shoot regeneration ability. In the transgenic, expression of the above shoot meristem-specific genes was elevated, which is consistent with the improved shoot regeneration. In contrast, the ARF10 deficient knockout mutant produced fewer regenerated shoot. However, overexpressors of ARF10 were only marginally more efficient than the wild type with the respect to shoot regeneration. Our observation strongly supports that proper shoot regeneration from in vitro cultured cells requires the miR160-directed negative influence of ARF10. The enhanced expression of ARF10 is likely to have contributed to the improved regeneration ability.     

Shoot bending promotes flower bud formation by miRNA‐mediated regulation in apple (Malus domestica Borkh.)

Flower induction in apple (Malus domestica Borkh.) trees plays an important life cycle role, but young trees produce fewer and inferior quality flower buds. Therefore, shoot bending has become an important cultural practice, significantly promoting the capacity to develop more flower buds during the growing seasons. Additionally, microRNAs (miRNAs) play essential roles in plant growth, flower induction and stress responses. In this study, we identified miRNAs potentially involved in the regulation of bud growth, and flower induction and development, as well as in the response to shoot bending. Of the 195 miRNAs identified, 137 were novel miRNAs. The miRNA expression profiles revealed that the expression levels of 68 and 27 known miRNAs were down‐regulated and up‐regulated, respectively, in response to shoot bending, and that the 31 differentially expressed novel miRNAs between them formed five major clusters. Additionally, a complex regulatory network associated with auxin, cytokinin, abscisic acid (ABA) and gibberellic acid (GA) plays important roles in cell division, bud growth and flower induction, in which related miRNAs and targets mediated regulation. Among them, miR396, 160, 393, and their targets associated with AUX, miR159, 319, 164, and their targets associated with ABA and GA, and flowering‐related miRNAs and genes, regulate bud growth and flower bud formation in response to shoot bending. Meanwhile, the flowering genes had significantly higher expression levels during shoot bending, suggesting that they are involved in this regulatory process. This study provides a framework for the future analysis of miRNAs associated with multiple hormones and their roles in the regulation of bud growth, and flower induction and formation in response to shoot bending in apple trees.     

Redundant and specific roles of individual MIR172 genes in plant development

Evolutionarily conserved microRNAs (miRNAs) usually have high copy numbers in the genome. The redundant and specific roles of each member of a multimember miRNA gene family are poorly understood. Previous studies have shown that the miR156-SPL-miR172 axis constitutes a signaling cascade in regulating plant developmental transitions. Here, we report the feasibility and utility of CRISPR-Cas9 technology to investigate the functions of all 5 MIR172 family members in Arabidopsis. We show that an Arabidopsis plant devoid of miR172 is viable, although it displays pleiotropic morphological defects. MIR172 family members exhibit distinct expression pattern and exert functional specificity in regulating meristem size, trichome initiation, stem elongation, shoot branching, and floral competence. In particular, we find that the miR156-SPL-miR172 cascade is bifurcated into specific flowering responses by matching pairs of coexpressed SPL and MIR172 genes in different tissues. Our results thus highlight the spatiotemporal changes in gene expression that underlie evolutionary novelties of a miRNA gene family in nature. The expansion of MIR172 genes in the Arabidopsis genome provides molecular substrates for the integration of diverse floral inductive cues, which ensures that plants flower at the optimal time to maximize seed yields.

This study uses CRISPR-Cas9 technology to investigate the functions of all five miR172 genes in Arabidopsis, finding that miRNA172 family members exhibit distinct expression pattern and exert functional specificity in regulating meristem size, trichome initiation, stem elongation, shoot branching and floral competence.     

高表达miR396小分子导致拟南芥花柱头弯曲

MicroRNAs（miRNAs）是大小约21个碱基、内源、非编码的小分子RNA。以拟南芥（Arabidopsis thaliana）miR396小分子为研究对象,分别克隆到了miR396小分子的两个前体（MIR396a,MIR396b）,得到了转基因植株。通过转基因植株的遗传学研究发现,高表达miR396小分子导致转基因拟南芥的花柱头弯曲。花柱头的弯曲影响了角果的正常发育。另外,Northern杂交结果表明转基因拟南芥花部位的miR396及其前体的表达量与对照相比显著增加。这些结果表明高表达miR396小分子可以导致拟南芥花柱头弯曲。     

Analysis of gradient-like expression of miR167 in Arabidopsis thaliana embryonic tissue

MicroRNAs (miRNAs) constitute a class of small non-coding endogenous RNAs that regulate gene expression by targeting mRNAs for degradation or translational repression. MiRNAs are intensively investigated and they have been found to be a pivotal component of developmental regulation processes. Recent studies showed the non-cell autonomous function of several miRNAs. We analyzed the accumulation pattern of selected miRNAs in Arabidopsis thaliana embryonic tissues. The majority of the investigated miRNAs showed uniform accumulation across the embryo suggesting their possible role at this developmental stage. In the case of miR167 however, we detected a gradient-like expression profile which in earlier studies has been considered to be the hallmark of the non-cell autonomous activity of miRNAs. Using reporter assay we analyzed the expression patterns of the four MIR167 precursor genes. We found that two of the precursor genes, MIR167A and MIR167B, also showed an overlapping gradient-like expression patterns in the embryo. These data indicate that in addition to non-cell autonomous activity of some miRNAs, the gradient-like expression patterns can be generated also by the specific expression characteristic of miRNA precursor genes.     

Control of root cap formation by MicroRNA-targeted auxin response factors in Arabidopsis

The plant root cap mediates the direction of root tip growth and protects internal cells. Root cap cells are continuously produced from distal stem cells, and the phytohormone auxin provides position information for root distal organization. Here, we identify the Arabidopsis thaliana auxin response factors ARF10 and ARF16, targeted by microRNA160 (miR160), as the controller of root cap cell formation. The Pro(35S):MIR160 plants, in which the expression of ARF10 and ARF16 is repressed, and the arf10-2 arf16-2 double mutants display the same root tip defect, with uncontrolled cell division and blocked cell differentiation in the root distal region and show a tumor-like root apex and loss of gravity-sensing. ARF10 and ARF16 play a role in restricting stem cell niche and promoting columella cell differentiation; although functionally redundant, the two ARFs are indispensable for root cap development, and the auxin signal cannot bypass them to initiate columella cell production. In root, auxin and miR160 regulate the expression of ARF10 and ARF16 genes independently, generating a pattern consistent with root cap development. We further demonstrate that miR160-uncoupled production of ARF16 exerts pleiotropic effects on plant phenotypes, and miR160 plays an essential role in regulating Arabidopsis development and growth.     

Computational identification of conserved microRNAs and their putative targets in the Hypericum perforatum L. flower transcriptome

MicroRNAs (miRNAs) have recently emerged as important regulators of gene expression in plants. Many miRNA families and their targets have been extensively studied in model species and major crops. We have characterized mature miRNAs along with their precursors and potential targets in Hypericum to generate a comprehensive list of conserved miRNA families and to investigate the regulatory role of selected miRNAs in biological processes that occur in the flower. St. John’s wort (Hypericum perforatum L., 2n = 4x = 32), a medicinal plant that produces pharmaceutically important metabolites with therapeutic activities, was chosen because it is regarded as an attractive model system for the study of apomixis. A computational in silico prediction of structure, in combination with an in vitro validation, allowed us to identify 7 pre-miRNAs, including miR156, miR166, miR390, miR394, miR396, and miR414. We demonstrated that H. perforatum flowers share highly conserved miRNAs and that these miRNAs potentially target dozens of genes with a wide range of molecular functions, including metabolism, response to stress, flower development, and plant reproduction. Our analysis paves the way toward identifying flower-specific miRNAs that may differentiate the sexual and apomictic reproductive pathways.