MiR160 and miR166/165 Contribute to the LEC2-Mediated Auxin Response Involved in the Somatic Embryogenesis Induction in Arabidopsis.
ABSTRACT: MicroRNAs are non-coding small RNA molecules that are involved in the post-transcriptional regulation of the genes that control various developmental processes in plants, including zygotic embryogenesis (ZE). miRNAs are also believed to regulate somatic embryogenesis (SE), a counterpart of the ZE that is induced in vitro in plant somatic cells. However, the roles of specific miRNAs in the regulation of the genes involved in SE, in particular those encoding transcription factors (TFs) with an essential function during SE including LEAFY COTYLEDON2 (LEC2), remain mostly unknown. The aim of the study was to reveal the function of miR165/166 and miR160 in the LEC2-controlled pathway of SE that is induced in in vitro cultured Arabidopsis explants.In ZE, miR165/166 controls the PHABULOSA/PHAVOLUTA (PHB/PHV) genes, which are the positive regulators of LEC2, while miR160 targets the AUXIN RESPONSE FACTORS (ARF10, ARF16, ARF17) that control the auxin signaling pathway, which plays key role in LEC2-mediated SE. We found that a deregulated expression/function of miR165/166 and miR160 resulted in a significant accumulation of auxin in the cultured explants and the spontaneous formation of somatic embryos. Our results show that miR165/166 might contribute to SE induction via targeting PHB, a positive regulator of LEC2 that controls embryogenic induction via activation of auxin biosynthesis pathway (Wójcikowska et al., 2013). Similar to miR165/166, miR160 was indicated to control SE induction through auxin-related pathways and the negative impact of miR160 on ARF10/ARF16/ARF17 was shown in an embryogenic culture. Altogether, the results suggest that the miR165/166- and miR160-node contribute to the LEC2-mediated auxin-related pathway of embryogenic transition that is induced in the somatic cells of Arabidopsis. A model summarizing the suggested regulatory interactions between the miR165/166-PHB and miR160-ARF10/ARF16/ARF17 nodes that control SE induction in Arabidopsis was proposed.
Project description:Global warming is causing a negative impact on plant growth and adversely impacts on crop yield. MicroRNAs (miRNAs) are critical in regulating the expression of genes involved in plant development as well as defense responses. The effects of miRNAs on heat-stressed Arabidopsis warrants further investigation. Heat stress increased the expression of miR160 and its precursors but considerably reduced that of its targets, ARF10, ARF16, and ARF17. To study the roles of miR160 during heat stress, transgenic Arabidopsis plants overexpressing miR160 precursor a (160OE) and artificial miR160 (MIM160), which mimics an inhibitor of miR160, were created. T-DNA insertion mutants of miR160 targets were also used to examine their tolerances to heat stress. Results presented that overexpressing miR160 improved seed germination and seedling survival under heat stress. The lengths of hypocotyl elongation and rachis were also longer in 160OE than the wild-type (WT) plants under heat stress. Interestingly, MIM160 plants showed worse adaption to heat. In addition, arf10, arf16, and arf17 mutants presented similar phenotypes to 160OE under heat stress to advance abilities of thermotolerance. Moreover, transcriptome and qRT-PCR analyses revealed that HSP17.6A, HSP17.6II, HSP21, and HSP70B expression levels were regulated by heat in 160OE, MIM160, arf10, arf16, and arf17 plants. Hence, miR160 altered the expression of the heat shock proteins and plant development to allow plants to survive heat stress.
Project description:MicroRNAs (miRNAs) are a class of non-coding RNAs that play important roles in plant development and abiotic stresses. To date, studies have mainly focused on the roles of individual miRNAs, however, a few have addressed the interactions among multiple miRNAs. In this study, we investigated the interplay and regulatory circuit between miR160 and miR165/166 and its effect on leaf development and drought tolerance in Arabidopsis using Short Tandem Target Mimic (STTM). By crossing STTM160 Arabidopsis with STTM165/166, we successfully generated a double mutant of miR160 and miR165/166. The double mutant plants exhibited a series of compromised phenotypes in leaf development and drought tolerance in comparison to phenotypic alterations in the single STTM lines. RNA-seq and qRT-PCR analyses suggested that the expression levels of auxin and ABA signaling genes in the STTM-directed double mutant were compromised compared to the two single mutants. Our results also suggested that miR160-directed regulation of auxin response factors (ARFs) contribute to leaf development via auxin signaling genes, whereas miR165/166- mediated HD-ZIP IIIs regulation confers drought tolerance through ABA signaling. Our studies further indicated that ARFs and HD-ZIP IIIs may play opposite roles in the regulation of leaf development and drought tolerance that can be further applied to other crops for agronomic traits improvement.
Project description:KEY MESSAGE:Extensive modulation of numerous ARF transcripts in the embryogenic culture of Arabidopsis indicates a substantial role of auxin signaling in the mechanism of somatic embryogenesis induction. Somatic embryogenesis (SE) is induced by auxin in plants and auxin signaling is considered to play a key role in the molecular mechanism that controls the embryogenic transition of plant somatic cells. Accordingly, the expression of AUXIN RESPONSE FACTOR (ARF) genes in embryogenic culture of Arabidopsis was analyzed. The study revealed that 14 of the 22 ARFs were transcribed during SE in Arabidopsis. RT-qPCR analysis indicated that the expression of six ARFs (ARF5, ARF6, ARF8, ARF10, ARF16, and ARF17) was significantly up-regulated, whereas five other genes (ARF1, ARF2, ARF3, ARF11, and ARF18) were substantially down-regulated in the SE-induced explants. The activity of ARFs during SE was also monitored with GFP reporter lines and the ARFs that were expressed in areas of the explants engaged in SE induction were detected. A functional test of ARFs transcribed during SE was performed and the embryogenic potential of the arf mutants and overexpressor lines was evaluated. ARFs with a significantly modulated expression during SE coupled with an impaired embryogenic response of the relevant mutant and/or overexpressor line, including ARF1, ARF2, ARF3, ARF5, ARF6, ARF8, and ARF11 were indicated as possibly being involved in SE induction. The study provides evidence that embryogenic induction strongly depends on ARFs, which are key regulators of the auxin signaling. Some clues on the possible functions of the candidate ARFs, especially ARF5, in the mechanism of embryogenic transition are discussed. The results provide guidelines for further research on the auxin-related functional genomics of SE and the developmental plasticity of somatic cells.
Project description:MicroRNA160 plays a critical role in plant development by negatively regulating the auxin response factors ARF10, -16, and -17. However, the ways in which miR160 expression is regulated at the transcriptional level, and how miR160 interacts with its targets during plant embryo development, remain unknown. Here, we studied the regulatory relationships among endogenous target mimics (eTMs), and miR160 and its targets, and their involvement in hormone signaling and somatic embryogenesis (SE) in Dimocarpus longan. We identified miR160 family members and isolated the miR160 precursor, primary transcript, and promoter. The promoter contained cis-acting elements responsive to stimuli such as light, abscisic acid, salicylic acid (SA) and heat stress. The pri-miR160 was down-regulated in response to SA but up-regulated by gibberellic acid, ethylene, and methyl jasmonate treatment, suggesting that pri-miR160 was associated with hormone transduction. Dlo-miR160a, -a(?) and -d(?) reached expression peaks in torpedo-shaped embryos, globular embryos and cotyledonary embryos, respectively, but were barely detectable in friable-embryogenic callus. This suggests that they have expression-related and functional diversity, especially during the middle and later developmental stages of SE. Four potential eTMs for miR160 were identified. Two of them, glucan endo-1,3-beta- glucosidase-like protein 2-like and calpain-type cysteine protease DEK1, were confirmed to control the corresponding dlo-miR160a(?) expression level. This suggests that they may function to abolish the binding between dlo-miR160a(?) and its targets. These two eTMs also participated in 2,4-D and ABA signal transduction. DlARF10, -16, and -17 targeting by dlo-miR160a was confirmed; their expression levels were higher in friable-embryogenic callus and incomplete compact pro-embryogenic cultures and responded to 2,4-D, suggesting they may play a major role in the early stages of longan SE dependent on 2,4-D. The eTMs, miR160, and ARF10, -16, and -17 exhibited tissue specificity in 'Sijimi' longan vegetative and reproductive organs, but were not significant negatively correlated. These results provide insights into the possible role of the eTM-miR160-ARF10-16-17 pathway in longan somatic embryo development.
Project description:The stem cell niche in the root meristem is critical for the development of the plant root system. The plant hormone auxin acts as a versatile trigger in many developmental processes, including the regulation of root growth, but its role in the control of the stem cell activity remains largely unclear. Here we show that local auxin levels, determined by biosynthesis and intercellular transport, mediate maintenance or differentiation of distal stem cells in the Arabidopsis thaliana roots. Genetic analysis shows that auxin acts upstream of the major regulators of the stem cell activity, the homeodomain transcription factor WOX5, and the AP-2 transcription factor PLETHORA. Auxin signaling for differentiation of distal stem cells requires the transcriptional repressor IAA17/AXR3 as well as the ARF10 and ARF16 auxin response factors. ARF10 and ARF16 activities repress the WOX5 transcription and restrict it to the quiescent center, where WOX5, in turn, is needed for the activity of PLETHORA. Our investigations reveal that long-distance auxin signals act upstream of the short-range network of transcriptional factors to mediate the differentiation of distal stem cells in roots.
Project description:The LEAFY COTYLEDON2 (LEC2) transcription factor with a plant-specific B3 domain plays a central role in zygotic and somatic embryogenesis (SE). LEC2 overexpression induced in planta leads to spontaneous somatic embryo formation, but impairs the embryogenic response of explants cultured in vitro under auxin treatment. The auxin-related functions of LEC2 appear during SE induction, and the aim of the present study was to gain further insights into this phenomenon. To this end, the effect of LEC2 overexpression on the morphogenic responses of Arabidopsis explants cultured in vitro under different auxin treatments was evaluated. The expression profiles of the auxin biosynthesis genes were analysed in embryogenic cultures with respect to LEC2 activity. The results showed that LEC2 overexpression severely modifies the requirement of cultured explants for an exogenous auxin concentration at a level that is effective in SE induction and suggested an increase in the auxin content in 35S::LEC2-GR transgenic explants. The assumption of an LEC2 promoted increase in endogenous auxin in cultured explants was further supported by the expression profiling of the genes involved in auxin biosynthesis. The analysis indicated that YUCCAs and TAA1, working in the IPA-YUC auxin biosynthesis pathway, are associated with SE induction, and that the expression of three YUCCA genes (YUC1, YUC4 and YUC10) is associated with LEC2 activity. The results also suggest that the IAOx-mediated auxin biosynthesis pathway involving ATR1/MYB34 and CYP79B2 does not seem to be involved in SE induction. We conclude that de novo auxin production via the tryptophan-dependent IPA-YUC auxin biosynthesis pathway is implicated in SE induction, and that LEC2 plays a key role in this mechanism.
Project description: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.
Project description:7-day-old seedlings were treated with heat stress at 44oC for 30 min. Total RNAs were isolated for library construction and analyzed by RNA-seq via Solexa platform. Raw sequences were obtained from the Illumina Pipeline software bcl2fastq v2.0 and expected to generate 20M (million reads or Gb) per sample. Overall design: Examination of transcriptome expression in WT, 160OE, arf10, arf16, and arf17 treating with/without heat stress
Project description:Callose synthesis is critical for the formation of the pollen wall pattern. CalS5 is thought to be the major synthethase for the callose wall. In the Arabidopsis anther, ARF17 regulates the expression of CalS5 and is the target of miR160. Plants expressing miR160-resistant ARF17 (35S:5mARF17 lines) with increased ARF17 mRNA levels display male sterility. Here we report a zinc finger family gene, AtTTP, which is involved in miR160 maturation and callose synthesis in Arabidopsis. AtTTP is expressed in microsporocytes, tetrads and tapetal cells in the anther. Over-expression lines of AtTTP (AtTTP-OE line) exhibited reduced male fertility. CalS5 expression was tremendously reduced and the tetrad callose wall became much thinner in the AtTTP-OE line. Northern blotting hybridization and quantitative RT-PCR analysis revealed that miR160 was decreased, while the expression of ARF17 was increased in the AtTTP-OE line. Based on these results, we propose that AtTTP associates with miR160 in order to regulate the ARF17 expression needed for callose synthesis and pollen wall formation.
Project description:To reveal how ARF10 and ARF16 regulate Al-induced root-growth inhibition, a transcriptome analysis was carried out by comparing without and with Al-exposed arf10/16 double mutant line and WT through RNA sequencing. The present transcriptomic analysis has revealed that many of the differentially transcribed genes associated with cell wall modification were regulated by transcription factors ARF10 and ARF16. The implication is that the auxin-regulated Al-induced inhibition of root growth arises from auxin signalling-regulated cell wall structure or component modification. Overall design: Total RNA obtained from control (non-treated roots) and Al-exposed roots. Six day old plants were treated with and without Al treatment for 24 h, then total RNA was isolated using the RNeasy® Plant Mini Kit (QIAGEN), and then Illumina sequencing was performed.