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© 2005 American Society of Plant Biologists
MicroRNAs Regulate Auxin Homeostasis and Plant Developmentneckardt{at}aspb.org The phytohormone auxin (principally indole-3-acetic acid [IAA]) is a key regulator of cell expansion and division that plays numerous roles in plant growth and development, including stem elongation, phototropic and gravitropic responses, apical dominance, and lateral and adventitious root formation. Auxin can stimulate or inhibit cell growth depending on its concentration and location within the plant, and its interactions with other hormone signaling pathways. In stems and coleoptiles, applied auxin induces cell elongation at low (µM) concentrations but inhibits elongation at higher (mM) concentrations. Auxin produced in the shoot apex inhibits the formation of lateral buds (apical dominance), but transported to the hypocotyls and root it promotes the production of adventitious and lateral roots, respectively. It has also been found to play a role in morphogenesis and patterning in leaves and floral organs. Normal plant development thus involves carefully controlled fluctuations in auxin biosynthesis, transport, accumulation, and degradation, which fine-tune auxin concentrations in specific tissues at specific stages of development to achieve the appropriate response.
In the past decade, there has been a growing understanding of the importance of protein degradation via the ubiquitin-proteasome pathway in regulating developmental processes and maintaining cellular homeostasis in eukaryotic organisms, and this pathway has been found to play a key role in auxin signaling in higher plants (Moon et al., 2004 The effects of auxin on plant development are mediated by several transcription factor families, including the auxin response factors (ARFs) and NAC-domain transcription factors. Sorin et al. (pages 1343–1359) show that ARGONAUTE1 (AGO1), a key player in microRNA pathways, regulates auxin-induced adventitious root formation associated with its effect on the expression of AUXIN RESPONSE FACTOR17 (ARF17) and auxin-inducible GH3 genes that are presumed targets of ARF17. Meanwhile, Mallory et al. (pages 1360–1375) show that plants expressing a form of ARF17 that is resistant to transcript cleavage mediated by the microRNA miR160 produce high levels of ARF17 mRNA and have altered accumulation of GH3-like mRNAs associated with numerous dramatic growth defects. Finally, Guo et al. (pages 1376–1386) show that miR164-directed cleavage of NAC1 mRNA affects auxin regulation of lateral root development and suggest a model for how microRNA-mediated regulation may function in maintaining auxin homeostasis.
MicroRNAs are endogenously encoded  22 nucleotide RNAs that target complementary mRNA transcripts for cleavage or translational repression via the RNA-induced silencing complex (RISC) (reviewed in Bartel, 2004 ). In plants, microRNAs are processed from hairpin precursor RNAs by an RNase III endonuclease known as DICER-LIKE1 (Park et al., 2002; Reinhart et al., 2002). The current model postulates that mature microRNAs are incorporated into the RISC, bind target mRNAs based on complementarity, and guide cleavage of mRNA targets with perfect or near perfect complementarity and translational repression of targets with lower complementarity (Bartel, 2004). The translational repression mechanism is common in animals but does not appear to play a major role in plants. The identity and activities of RISC components are incompletely understood, but in all eukaryotes examined, a member of the AGO protein family is a principal RISC component (Hammond et al., 2001).
Arabidopsis AGO1 is the founding member of this family of proteins. Homozygous ago1-1 mutants show a severely abnormal phenotype characterized by unexpanded, pointed cotyledons and narrow, almost succulent, rosette leaves (Bohmert et al., 1998 Analysis of auxin content and the rate of auxin biosynthesis in ago1 mutants relative to superroot2 (sur2) mutants (which overproduce auxin and develop numerous adventitious roots on the hypocotyls) and in ago1 sur2 double mutants suggested that AGO1 influences the overall regulation of auxin homeostasis in the hypocotyl. The investigation of several auxin-inducible genes revealed that the expression of several GH3 genes in particular was downregulated in ago1 mutant hypocotyls. Finally, the authors found that ARF17 was significantly overexpressed in mutant hypocotyls. The authors suggest a model in which ARF17 negatively regulates GH3 gene expression and adventitious root formation in Arabidopsis, through its regulation by AGO1-dependent microRNA-mediated repression.
The importance of microRNA regulation of ARF17 is also revealed by the work of Mallory et al. Rhoades et al. (2002)
The work of Sorin et al. and Mallory et al. suggest that AGO1 functions with miR160 to repress ARF17, to allow proper auxin homeostasis and development. In a related article, Guo et al. found that miR164-directed cleavage of NAC1 mRNA downregulates auxin signals for lateral root development. Xie et al. (2000) previously showed that NAC1 functions in auxin induction of lateral root development, and Rhoades et al. (2002) identified NAC1 as a potential target of microRNA regulation by miR164. Guo et al. confirmed that NAC1 mRNA cleavage occurs in vivo and is directed by miR164. Similar to the approach of Mallory et al., they created transgenic plants expressing a cleavage-resistant form of NAC1 mRNA. These plants accumulate NAC1 mRNA and significantly increase initiation of lateral roots, suggesting that microRNA-mediated cleavage is an important negative regulator of NAC1 in wild-type plants. Xie et al. (2000) showed that NAC1 is a transcriptional activator of the auxin-responsive genes DBP, which encodes another DNA binding protein of unknown function, and AIR3, which encodes a protein with homology to subtilisin-like proteases, and NAC1 activity is associated with lateral root production.
Also in this issue, Inukai et al. (pages 1387–1396) show that CROWN ROOTLESS1 (CRL1) is essential for crown root formation in rice and that it is a target of an ARF in the auxin signaling pathway. This work raises the intriguing question of whether the rice ARF (and, thus, its target CRL1) is subject to microRNA-mediated regulation. Although Arabidopsis is capable of forming adventitious roots, they do not play an important role in normal plant development in the species, or indeed in many other dicots. By contrast, rice and other monocots produce numerous crown roots, which are a type of adventitious root that is one of the dominant root types of cereals. The regulation of adventitious root formation in rice therefore is of considerable interest. Jones-Rhoades and Bartel (2004)
MicroRNAs regulate many genes, and the readout is complex. For example, Sorin et al. found that disruption of AGO1 resulted in overproduction of ARF17 and reduced GH3 expression in the hypocotyl. Mallory et al. similarly found that expression of microRNA-resistant ARF17 was associated with overproduction of ARF17 mRNA and reductions in GH3 expression, and although altered phenotypes were seen in root as well as shoot development, auxin responsiveness was maintained in roots and adventitious rooting was not reported. The phenotypic differences in the two studies are likely due to a general impairment of all microRNAs in the ago1 mutant (Vaucheret et al., 2004 ) versus the misregulation of a single target in the Mallory et al. study. It is reasonable to expect that ago1 mutant phenotypes result from the collective misregulation of many targets, including not only ARF17, but also ARF6, ARF8, ARF10, ARF16, NAC1, and TIR1, to name a few. However, there are intriguing similarities between ago1 mutants and miR160-resistant ARF17 plants, suggesting that ARF17 overexpression contributes to a subset of the ago1 phenotypes. Together, these studies suggest that microRNA-mediated regulation of ARF17 is a major facet of auxin homeostasis and plant development, which functions at least in part via an effect on GH3 gene expression.
It is notable that the results of Sorin et al. suggest that AGO1 influences auxin homeostasis primarily in the apical part of the plant and not in the root (i.e., ago1 mutants are affected in adventitious root but not lateral root formation) and that a target of AGO1 activity in the hypocotyl is ARF17. On the other hand, Guo et al. show that microRNA-mediated regulation of NAC1 is part of the auxin response pathway in the root, affecting lateral root development. Because AGO1 is involved in the microRNA regulation of many targets (Vaucheret et al., 2004
All three reports (Guo et al., Mallory et al., and Sorin et al.) provide evidence that microRNAs regulate auxin homeostasis in Arabidopsis. The evidence for this is severalfold. First, and most obviously, disruption of AGO1 or of the microRNA-mediated cleavage of NAC1 target mRNA caused dramatic effects on auxin induction of adventitious and lateral root formation, respectively. Second, the GH3 genes that are the putative targets of ARF17 encode auxin-conjugating proteins (Staswick et al., 2005 ), and regulation of these genes may provide an important mechanism for controlling levels of free IAA (active auxin). ARF17 may act as a repressor of some GH3 genes because overaccumulation of ARF17 mRNA is correlated with reduced levels of some GH3 mRNAs. Reducing microRNA-mediated cleavage of ARF17 mRNA therefore might be expected to increase levels of free IAA, as repression of GH3 transcription would be increased, reducing the level of the corresponding auxin-conjugating proteins and resulting in less conjugation of IAA. However, Sorin et al. measured a significant reduction in endogenous levels of not only conjugated but also free IAA in ago1 mutant plants. They noted that auxin biosynthesis was slightly lower in the mutant relative to wild-type plants, which could be due to a direct effect of AGO1 on regulation of auxin biosynthesis or an indirect effect of disrupting overall auxin homeostasis. Both Mallory et al. and Guo et al. also investigated the question of whether auxin affects microRNA levels. Mallory et al. isolated total RNA from wild-type seedlings at 0, 0.3, 2, and 24 h after treatment with 10 µM IAA and could see no appreciable effect of the IAA treatment on levels of the miR160, miR164, or miR167 microRNAs (all of which have been implicated in auxin signaling). In addition, levels of ARF17 mRNA and the ARF17 mRNA cleavage product did not appear to be affected by auxin treatment. Thus, it is clear that levels of these microRNAs are not dramatically responding to IAA as do many of the mRNAs encoding other repressors acting in auxin responses, such as the Aux/IAA transcriptional repressors and the GH3-like auxin conjugating enzymes. Mallory et al. concluded that "regulation by these three miRNAs may instead be needed to set components of the auxin-response machinery to proper levels so that tissues can respond appropriately to auxin."
Guo et al. also measured auxin induction of expression of miR164 in wild-type plants and were able to detect a subtle difference in levels after synthetic auxin treatment. NAC1 is an early auxin response gene, and an increase in NAC1 mRNA was seen within 30 min after treatment with the synthetic auxin NAA at a concentration of 2 µM. Similar to the results of Mallory et al. with IAA, the authors did not observe any change in miR164 levels under these conditions. However, Guo et al. report a consistent These reports provide significant new information on microRNA-mediated regulation of plant development and help to paint a clearer picture of the nature of auxin homeostasis—as such, they constitute required reading for all students of plant developmental biology!
Bartel, D.P. (2004). MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell 116, 281–297.[CrossRef][Web of Science][Medline] Bohmert, K., Camus, I., Bellini, C., Bouchez, D., Caboche, M., and Benning, C. (1998). AGO1 defines a novel locus of Arabidopsis controlling leaf development. EMBO J. 17, 170–180.[CrossRef][Web of Science][Medline]
Carmell, M.A., Xuan, Z., Zhang, M.Q., and Hannon, G.J. (2002). The Argonaute family: Tentacles that reach into RNAi, developmental control, stem cell maintenance, and tumorigenesis. Genes Dev. 16, 2733–2742.
Guo, H.-S., Xie, Q., Fei, J.-F., and Chua, N.-H. (2005). MicroRNA directs mRNA cleavage of the transcription factor NAC1 to downregulate auxin signals for Arabidopsis lateral root development. Plant Cell 17, 1376–1386.
Hammond, S.M., Boettcher, S., Caudy, A.A., Kobayashi, R., and Hannon, G.J. (2001). Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 293, 1146–1150.
Inukai, Y., Sakamoto, T., Ueguchi-Tanaka, M., Shibata, Y., Gomi, K., Umemura, I., Hasegawa, Y., Ashikari, M., Kitano, H., and Matsuoka, M. (2005). Crown rootless1, which is essential for crown root formation in rice, is a target of an AUXIN RESPONSE FACTOR in auxin signaling. Plant Cell 17, 1387–1396. Jones-Rhoades, M.W., and Bartel, D.P. (2004). Computational identification of plant microRNAs and their targets, including a stress-induced miRNA. Mol. Cell 14, 787–799.[CrossRef][Web of Science][Medline]
Mallory, A.C., Bartel, D.P., and Bartel, B. (2005). MicroRNA-directed regulation of Arabidopsis AUXIN RESPONSE FACTOR17 is essential for proper development and modulates expression of early auxin response genes. Plant Cell 17, 1360–1375.
Moon, J., Parry, G., and Estelle, M. (2004). The ubiquitin-proteasome pathway and plant development. Plant Cell 16, 3181–3195. Park, W., Li, J., Song, R., Messing, J., and Chen, X. (2002). CARPEL FACTORY, a Dicer homolog, and HEN1, a novel protein, act in microRNA metabolism in Arabidopsis thaliana. Curr. Biol. 12, 1484–1495.[CrossRef][Web of Science][Medline]
Reinhart, B.J., Weinstein, E.G., Rhoades, M.W., Bartel, B., and Bartel, D.P. (2002). MicroRNAs in plants. Genes Dev. 16, 1616–1626. Rhoades, M., Reinhart, B., Lim, L., Burge, C., Bartel, B., and Bartel, D.P. (2002). Prediction of plant microRNA targets. Cell 110, 513–520.[CrossRef][Web of Science][Medline]
Sorin, C., Bussell, J.D., Camus, I., Ljung, K., Kowalczyk, M., Geiss, G., McKhann, H., Garcion, C., Vaucheret, H., Sandberg, G., and Bellini, C. (2005). Auxin and light control of adventitious rooting in Arabidopsis require ARGONAUTE1. Plant Cell 17, 1343–1359.
Staswick, P.E., Serban, B., Rowe, M., Tiryaki, I., Maldonado, M.T., Maldonado, M.C., and Suza, W. (2005). Characterization of an Arabidopsis enzyme family that conjugates amino acids to indole-3-acetic acid. Plant Cell 17, 616–627.
Sunkar, R., Girke, T., Jain, P.K., and Zhu, J.-K. (2005). Cloning and characterization of microRNAs from rice. Plant Cell 17, 1397–1411.
Vaucheret, H., Vazquez, F., Crété, P., and Bartel, D.P. (2004). The action of ARGONAUTE1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant development. Genes Dev. 18, 1187–1197.
Xie, Q., Frugis, G., Colgan, D., and Chua, N.-H. (2000). Arabidopsis NAC1 transduces auxin signal downstream of TIR1 to promote lateral root development. Genes Dev. 14, 3024–3036. Related articles in Plant Cell:
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