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.     

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.     

HYL1 regulates the balance between adaxial and abaxial identity for leaf flattening via miRNA-mediated pathways

HYPONASTIC LEAVES1 (HYL1) is an important regulator of microRNA (miRNA) biogenesis. Incurvature of rosette leaves in loss-of-function mutants of HYL1 implicates the regulation of leaf flatness by HYL1 via miRNA pathways. Recent studies have identified jba-1D, jaw-1D, and oe-160c, the dominant mutants of MIR166g, MIR319a, and MIR160c genes, respectively, which display three types of leaf curvature. However, it remains unclear whether or how HYL1 controls leaf flatness through the pathways mediated by these miRNAs. To define which miRNAs and target genes are relevant to the hyl1 phenotype in terms of leaf incurvature, the effects of three mutated MIRNA genes and their targets on the direction and extent of leaf curvature in hyl1 mutants were examined. The genetic analysis shows that the hyl1 phenotype is strongly rescued by jba-1D, but not by jaw-1D or oe-160c, whereas the mutant phenotypes of jba-1D, jaw-1D, or oe-160c leaves are compromised by the hyl1 allele. Expression analysis indicates that reduced accumulation of miR166, rather than of miR319a or miR160, causes incurvature of hyl1 leaves, and that miR319a-targeted TCP3 positively regulates the adaxial identity gene PHABULOSA while miR160-targeted ARF16 negatively regulates the abaxial identity gene FILAMENTOUS FLOWER. In these cases, the direction and extent of leaf incurvature are associated with the expression ratio of adaxial to abaxial genes (adaxial to abaxial ratio). HYL1 regulates the balance between adaxial and abaxial identity and modulates leaf flatness by preventing leaf incurvature, wavy margins, and downward curvature. It is concluded that HYL1 monitors the roles of miR165/166, miR319a, and miR160 in leaf flattening through the relative activities of adaxial and abaxial identity genes, thus playing an essential role in leaf development.     

LNA mediated in situ hybridization of miR171 and miR397a in leaf and ambient root tissues revealed expressional homogeneity in response to shoot heat shock in Arabidopsis thaliana

MicroRNAs (miRNAs) are endogenous non-protein coding RNA molecules of approximately 21 nucleotides in length capable of modulating gene expression in animals and plants. The role of miRNA based gene regulation has been proved in several pathways including in plant growth, development and stress response. In this study miR171 and miR397a were tested for their expression pattern under different heat shock regimes in shoot and root tissues of Arabidopsis thaliana using Locked Nucleic Acid (LNA) mediated in situ hybridization. With an increase in temperature across 35 °C, 40 °C and 45 °C there was a corresponding increased up-regulation of miR171 in leaf tissues compared to ambient temperature. Similarly, an unambiguous elevated expression of miR171 within increase in duration of exposure at each temperature regime across 1 h, 2 h and 3 h was noticed in comparison to ambient control leaf tissue. On the other hand, miR397a, which expressed at ambient control conditions, got down-regulated both with increase in heat and exposure regime in leaf tissues. Both miRNAs expressed in control ambient root tissues. Maintaining the root zone temperature at ambient conditions, upon imposing heat shock regime to shoot system, miR171 recorded corresponding increased up-regulation as indicated by the intensity of in situ hybridization, while miR397a got down-regulated. Given the differential homogeneity in expression pattern of both miRNA in leaf and root tissues experiencing heat shock regimes, possibilities of movement of heat shock induced signals to root tissues seem to be obvious.     

MicroRNA directs mRNA cleavage of the transcription factor NAC1 to downregulate auxin signals for arabidopsis lateral root development

Although several plant microRNAs (miRNAs) have been shown to play a role in plant development, no phenotype has yet been associated with a reduction or loss of expression of any plant miRNA. Arabidopsis thaliana miR164 was predicted to target five NAM/ATAF/CUC (NAC) domain-encoding mRNAs, including NAC1, which transduces auxin signals for lateral root emergence. Here, we show that miR164 guides the cleavage of endogenous and transgenic NAC1 mRNA, producing 3'-specific fragments. Cleavage was blocked by NAC1 mutations that disrupt base pairing with miR164. Compared with wild-type plants, Arabidopsis mir164a and mir164b mutant plants expressed less miR164 and more NAC1 mRNA and produced more lateral roots. These mutant phenotypes can be complemented by expression of the appropriate MIR164a and MIR164b genomic sequences. By contrast, inducible expression of miR164 in wild-type plants led to decreased NAC1 mRNA levels and reduced lateral root emergence. Auxin induction of miR164 was mirrored by an increase in the NAC1 mRNA 3' fragment, which was not observed in the auxin-insensitive mutants auxin resistant1 (axr1-12), axr2-1, and transport inhibitor response1. Moreover, the cleavage-resistant form of NAC1 mRNA was unaffected by auxin treatment. Our results indicate that auxin induction of miR164 provides a homeostatic mechanism to clear NAC1 mRNA to downregulate auxin signals.     

A miRNA involved in phosphate-starvation response in Arabidopsis

Although microRNAs (miRNAs) have been documented to regulate development in plants and animals , the function of miRNAs in physiology is unclear. miR399 has multiple target sites in the 5' untranslated region (UTR) of a gene encoding a putative ubiquitin-conjugating enzyme (UBC) in Arabidopsis thaliana. We report here that miR399 was highly induced, whereas the target UBC mRNA was reduced by low-phosphate (Pi) stress. In transgenic plants with constitutive expression of miR399, UBC mRNA accumulation was suppressed even under high Pi. The expression of transgene UBC mRNA with 5' UTR miR399 target sites, but not the one without 5' UTR, was reduced under low-Pi condition. Furthermore, transgenic Arabidopsis plants with constitutive expression of miR399 accumulated more Pi than the wild-type, and transgenic plants expressing the UBC mRNA without 5' UTR (miRNA-deregulated) showed less inhibition of primary root growth and less induction of a Pi transporter gene by low-Pi stress than those of wild-type plants. We conclude that miR399 downregulates UBC mRNA accumulation by targeting the 5' UTR, and this regulation is important for plant responses to Pi starvation. The results suggest that miRNAs have functional roles for plants to cope with fluctuations in mineral-nutrient availability in the soil.     

Involvement of miR169 in the nitrogen-starvation responses in Arabidopsis

Recent studies have revealed that microRNAs (miRNAs) regulate plant adaptive responses to nutrient deprivation. However, the functional significance of miRNAs in adaptive responses to nitrogen (N) limitation remains to be explored. The Arabidopsis miR169 was strongly down-regulated, whereas its targets, NFYA (Nuclear Factor Y, subunit A) family members, were strongly induced by nitrogen N starvation. Analysis of the expression of miR169 precursors showed that MIR169a was substantially down-regulated in both roots and shoots by N starvation. Accumulation of the NFYA family members was suppressed in transgenic Arabidopsis with constitutive expression of MIR169a. Transgenic Arabidopsis plants overexpressing MIR169a accumulated less N and were more sensitive to N stress than the wild type. N sensitivity of 35S::MIR169a might be attributable to impaired uptake systems. These results provide evidence that miRNAs have functional roles in helping plants to cope with fluctuations in N availability in the soil.