The role of miR156/SPLs modules in Arabidopsis lateral root development

miR156 is an evolutionarily highly conserved miRNA in plants that defines an age‐dependent flowering pathway. The investigations thus far have largely, if not exclusively, confined to plant aerial organs. Root branching architecture is a major determinant of water and nutrients uptake for plants. We show here that MIR156 genes are differentially expressed in specific cells/tissues of lateral roots. Plants overexpressing miR156 produce more lateral roots whereas reducing miR156 levels leads to fewer lateral roots. We demonstrate that at least one representative from the three groups of miR156 targets SQUAMOSA PROMOTER BINDING PROTEIN‐LIKE (SPL) genes: SPL3, SPL9 and SPL10 are involved in the repression of lateral root growth, with SPL10 playing a dominant role. In addition, both MIR156 and SPLs are responsive to auxin signaling suggesting that miR156/SPL modules might be involved in the proper timing of the lateral root developmental progression. Collectively, these results unravel a role for miR156/SPLs modules in lateral root development in Arabidopsis.     

The microRNA miR171h modulates arbuscular mycorrhizal colonization of Medicago truncatula by targeting NSP2

Most land plants live symbiotically with arbuscular mycorrhizal fungi. Establishment of this symbiosis requires signals produced by both partners: strigolactones in root exudates stimulate pre‐symbiotic growth of the fungus, which releases lipochito‐oligosaccharides (Myc‐LCOs) that prepare the plant for symbiosis. Here, we have investigated the events downstream of this early signaling in the roots. We report that expression of miR171h, a microRNA that targets NSP2, is up‐regulated in the elongation zone of the root during colonization by Rhizophagus irregularis (formerly Glomus intraradices) and in response to Myc‐LCOs. Fungal colonization was much reduced by over‐expressing miR171h in roots, mimicking the phenotype of nsp2 mutants. Conversely, in plants expressing an NSP2 mRNA resistant to miR171h cleavage, fungal colonization was much increased and extended into the elongation zone of the roots. Finally, phylogenetic analyses revealed that miR171h regulation of NSP2 is probably conserved among mycotrophic plants. Our findings suggest a regulatory mechanism, triggered by Myc‐LCOs, that prevents over‐colonization of roots by arbuscular mycorrhizal fungi by a mechanism involving miRNA‐mediated negative regulation of NSP2.     

Improving rice tolerance to potassium deficiency by enhancing OsHAK16p:WOX11‐controlled root development

Potassium (K) deficiency in plants confines root growth and decreases root‐to‐shoot ratio, thus limiting root K acquisition in culture medium. A WUSCHEL‐related homeobox (WOX) gene, WOX11, has been reported as an integrator of auxin and cytokinin signalling that regulates root cell proliferation. Here, we report that ectopic expression of WOX11 gene driven by the promoter of OsHAK16 encoding a low‐K‐enhanced K transporter led to an extensive root system and adventitious roots and more effective tiller numbers in rice. The WOX11‐regulated root and shoot phenotypes in the OsHAK16p:WOX11 transgenic lines were supported by K‐deficiency‐enhanced expression of several RR genes encoding type‐A cytokinin‐responsive regulators, PIN genes encoding auxin transporters and Aux/IAA genes. In comparison with WT, the transgenic lines showed increases in root biomass, root activity and K concentrations in the whole plants, and higher soluble sugar concentrations in roots particularly under low K supply condition. The improvement of sugar partitioning to the roots by the expression of OsHAK16p:WOX11 was further indicated by increasing the expression of OsSUT1 and OsSUT4 genes in leaf blades and several OsMSTs genes in roots. Expression of OsHAK16p:WOX11 in the rice grown in moderate K‐deficient soil increased total K uptake by 72% and grain yield by 24%–32%. The results suggest that enlarging root growth and development by the expression of WOX11 in roots could provide a useful option for increasing K acquisition efficiency and cereal crop productivity in low K soil.     

A common miRNA160‐based mechanism regulates ovary patterning,floral organ abscission and lamina outgrowth in tomato

Plant microRNAs play vital roles in auxin signaling via the negative regulation of auxin response factors (ARFs). Studies have shown that targeting of ARF10/16/17 by miR160 is indispensable for various aspects of development, but its functions in the model crop tomato (Solanum lycopersicum) are unknown. Here we knocked down miR160 (sly–miR160) using a short tandem target mimic (STTM160), and investigated its roles in tomato development. Northern blot analysis showed that miR160 is abundant in developing ovaries. In line with this, its down‐regulation perturbed ovary patterning as indicated by the excessive elongation of the proximal ends of mutant ovaries and thinning of the placenta. Following fertilization, these morphological changes led to formation of elongated, pear‐shaped fruits reminiscent of those of the tomato ovate mutant. In addition, STTM160‐expressing plants displayed abnormal floral organ abscission, and produced leaves, sepals and petals with diminished blades, indicating a requirement for sly–miR160 for these auxin‐mediated processes. We found that sly–miR160 depletion was always associated with the up‐regulation of SlARF10A, SlARF10B and SlARF17, of which the expression of SlARF10A increased the most. Despite the sly–miR160 legitimate site of SlARF16A, its mRNA levels did not change in response to sly–miR160 down‐regulation, suggesting that it may be regulated by a mechanism other than mRNA cleavage. SlARF10A and SlARF17 were previously suggested to function as inhibiting ARFs. We propose that by adjusting the expression of a group of ARF repressors, of which SlARF10A is a primary target, sly–miR160 regulates auxin‐mediated ovary patterning as well as floral organ abscission and lateral organ lamina outgrowth.     

KITLG is a novel target of miR‐34c that is associated with the inhibition of growth and invasion in colorectal cancer cells

MiR‐34c is considered a potent tumour suppressor because of its negative regulation of multiple target mRNAs that are critically associated with tumorigenesis and metastasis. In the present study, we demonstrated a novel target of miR‐34c, KITLG, which has been implicated in colorectal cancer (CRC). First, we found a significant negative relationship between miR‐34c and KITLG mRNA expression levels in CRC cell lines, including HT‐29, HCT‐116, SW480 and SW620 CRC cell lines. In silico analysis predicted putative binding sites for miR‐34c in the 3′ untranslated region (3′UTR) of KITLG mRNA. A dual‐luciferase reporter assay further confirmed that KITLG is a direct target of miR‐34c. Then, the cell lines were infected with lentiviruses expressing miR‐34c or a miR‐34c specific inhibitor. Restoration of miR‐34c dramatically reduced the expression of KITLG mRNA and protein, while silencing of endogenous miR‐34c increased the expression of KITLG protein. The miR‐34c‐mediated down‐regulation of KITLG was associated with the suppression on proliferation, cellular transformation, migration and invasion of CRC cells, as well as the promotion on apoptosis. Knockdown of KITLG by its specific siRNA confirmed a critical role of KITLG down‐regulation for the tumour‐suppressive effects of miR‐34c in CRC cells. In conclusion, our results demonstrated that miR‐34c might interfere with KITLG‐related CRC and could be a novel molecular target for CRC patients.     

MicroRNA‐183‐5p is stress‐inducible and protects neurons against cell death in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the death of motor neurons. A fundamental pathogenesis of ALS is the prolonged cell stress in neurons, which is caused by either accumulation of protein aggregates or reactive oxygen species. However, the mechanistic link between stress sensing and cell death is unsettled. Here, we identify that miR‐183‐5p, a neuron‐enriched miRNA, couples stress sensing and cell death programming in ALS. miR‐183‐5p is immediately induced by hydrogen peroxide, tunicamycin or TNF‐α in neurons. The overexpression of miR‐183‐5p increases neuron survival under stress conditions, whereas its knockdown causes neuron death. miR‐183‐5p coordinates apoptosis and necroptosis pathways by directly targeting PDCD4 and RIPK3, and thus protects neurons against cell death under stress conditions. The consistent reduction of miR‐183‐5p in ALS patients and mouse models enhances the notion that miR‐183‐5p is a central regulator of motor neuron survival under stress conditions. Our study supplements current understanding of the mechanistic link between cell stress and death/survival, and provides novel targets for clinical interventions of ALS.     

Involvement of LeMRP,an ATP‐binding cassette transporter,in shikonin transport and biosynthesis in Lithospermum erythrorhizon

• Shikonin and its derivatives are important medicinal secondary metabolites accumulating in roots of Lithospermum erythrorhizon. Although some membrane proteins have been identified as transporters of secondary metabolites, the mechanisms underlying shikonin transport and accumulation in L. erythrorhizon cells still remain largely unknown.
• In this study, we isolated a cDNA encoding LeMRP, an ATP‐binding cassette transporter from L. erythrorhizon, and further investigated its functions in the transport and biosynthesis of shikonin using the yeast transformation and transgenic hairy root methods, respectively. Real‐time PCR was applied for expression analyses of LeMRP and shikonin biosynthetic enzyme genes.
• Functional analysis of LeMRP using the heterologous yeast cell expression system showed that LeMRP could be involved in shikonin transport. Transgenic hairy roots of L. erythrorhizon demonstrated that LeMRP overexpressing hairy roots produced more shikonin than the empty vector (EV) control. Real‐time PCR results revealed that the enhanced shikonin biosynthesis in the overexpression lines was mainly caused by highly up‐regulated expression of genes coding key enzymes (LePAL, HMGR, Le4CL and LePGT) involved in shikonin biosynthesis. Conversely, LeMRP RNAi decreased the accumulation of shikonin and effectively down‐regulated expression level of the above genes. Typical inhibitors of ABC proteins, such as azide and buthionine sulphoximine, dramatically inhibited accumulation of shikonin in hairy roots.
• Our findings provide evidence for the important direct or indirect role of LeMRP in transmembrane transport and biosynthesis of shikonin.

     

PeCHYR1, a ubiquitin E3 ligase from Populus euphratica,enhances drought tolerance via ABA‐induced stomatal closure by ROS production in Populus

Drought, a primary abiotic stress, seriously affects plant growth and productivity. Stomata play a vital role in regulating gas exchange and drought adaptation. However, limited knowledge exists of the molecular mechanisms underlying stomatal movement in trees. Here, PeCHYR1, a ubiquitin E3 ligase, was isolated from Populus euphratica, a model of stress adaptation in forest trees. PeCHYR1 was preferentially expressed in young leaves and was significantly induced by ABA (abscisic acid) and dehydration treatments. To study the potential biological functions of PeCHYR1, transgenic poplar 84K (Populus alba × Populus glandulosa) plants overexpressing PeCHYR1 were generated. PeCHYR1 overexpression significantly enhanced H₂O₂ production and reduced stomatal aperture. Transgenic lines exhibited increased sensitivity to exogenous ABA and greater drought tolerance than that of WT (wild‐type) controls. Moreover, up‐regulation of PeCHYR1 promoted stomatal closure and decreased transpiration, resulting in strongly elevated WUE (water use efficiency). When exposed to drought stress, transgenic poplar maintained higher photosynthetic activity and biomass accumulation. Taken together, these results suggest that PeCHYR1 plays a crucial role in enhancing drought tolerance via ABA‐induced stomatal closure caused by hydrogen peroxide (H₂O₂) production in transgenic poplar plants.     

microRNA159‐targeted SlGAMYB transcription factors are required for fruit set in tomato

The transition from flowering to fruit production, namely fruit set, is crucial to ensure successful sexual plant reproduction. Although studies have described the importance of hormones (i.e. auxin and gibberellins) in controlling fruit set after pollination and fertilization, the role of microRNA‐based regulation during ovary development and fruit set is still poorly understood. Here we show that the microRNA159/GAMYB1 and ‐2 pathway (the miR159/GAMYB1/2 module) is crucial for tomato ovule development and fruit set. MiR159 and SlGAMYBs were expressed in preanthesis ovaries, mainly in meristematic tissues, including developing ovules. SlMIR159‐overexpressing tomato cv. Micro‐Tom plants exhibited precocious fruit initiation and obligatory parthenocarpy, without modifying fruit shape. Histological analysis showed abnormal ovule development in such plants, which led to the formation of seedless fruits. SlGAMYB1/2 silencing in SlMIR159‐overexpressing plants resulted in misregulation of pathways associated with ovule and female gametophyte development and auxin signalling, including AINTEGUMENTA‐like genes and the miR167/SlARF8a module. Similarly to SlMIR159‐overexpressing plants, SlGAMYB1 was downregulated in ovaries of parthenocarpic mutants with altered responses to gibberellins and auxin. SlGAMYBs likely contribute to fruit initiation by modulating auxin and gibberellin responses, rather than their levels, during ovule and ovary development. Altogether, our results unveil a novel function for the miR159‐targeted SlGAMYBs in regulating an agronomically important trait, namely fruit set.     

Proteomic screening and identification of microRNA‐128 targets in glioma cells

Brain‐enriched miR‐128 is repressed in glioma cells, and could inhibit the proliferation of gliomas by targeting genes such as E2F3a and BMI1. To identify more targets of miR‐128 in glioblastoma multiforme, the pulse stable isotope labeling with amino acids in cell culture (pSILAC) technique was used to test its impact on whole protein synthesis in T98G glioma cells. We successfully identified 1897 proteins, of which 1459 proteins were quantified. Among them, 133 proteins were downregulated after the overexpression of miR‐128. Through predictions using various bioinformatics tools, 13 candidate target genes were chosen. A luciferase assay validated that 11 of 13 selected genes were potential targets of miR‐128, and a mutagenesis experiment confirmed CBFB, CORO1C, GLTP, HnRNPF, and TROVE2 as the target genes. Moreover, we observed that the expression of CORO1C, TROVE2, and HnRNPF were higher in glioma cell lines compared to normal brain tissues and presented a tendency toward downregulation after overexpression of miR‐128 in T98G cells. Furthermore, we have validated that CORO1C, TROVE2, and HnRNPF could inhibit glioma cell proliferation. In sum, our data showed that the integration of pSILAC and bioinformatics analysis was an efficient method for seeking the targets of miRNAs, and plentiful targets of miR‐128 were screened and laid the foundation for research into the miR‐128 regulation network.