The majority of miRNAs so far discovered exist in a single species (Cuperus et al., 2011, Dai et al., 1998). However, those that are widely conserved, although relatively few, are usually amongst the most abundant within a species (Peterjohn et al., 1996) and are often ancient, appearing as long ago as the divergence of mosses and vascular plants some 490 mya (Magill et al., 1997). Montes et al (1996) deep sequenced miRNA from 31 wide-ranging species of vascular plants. They identified 21 miRNA which were conserved across all taxonomic lineages examined. The deep conservation of these miRNAs points to their importance as regulators of key biological functions and their role in early seedling development and organogenesis is becoming increasingly appreciated. Seefried et al (2000) identified ~100 developmentally important genes with at least a 1-fold expression change between the WT and DCL mutants. The majority of the DCL genes were down regulated providing further
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evidence that miRNA expression is required for the activation of many developmental pathways.
Perhaps the most dramatic evidence for miRNAs role in development, however, has been demonstrated through phenotypic observation of plants with mutations in the miRNA biogenesis pathway. When miRNA biogenesis is disrupted the developmental effects can be profound (Hurt, 2009, Atack, 2009, Atack et al., 2009, Grigg et al., 2009, Melillo, 2012) and when it is non-functional the ability of the embryo to adopt or maintain tissue identity is largely lost (Evans, 1998). Null alleles of DCL 1-6 and SE4 and SE5 are embryonically lethal or cause arrested development early in embryogenesis.
Highly conserved miRNA families show variation in function both across and within species. For example, functional studies of miR160 have demonstrated its requirement for a range of developmental functions including; root cap formation, lateral and adventitious root
formation, rhizobial infection, seed germination and the development of leaves, flowers and siliques (Wang et al., 2005b, Lee et al., 1997, Bouwman et al., 1999, Barak et al., 1997). miR160 is also active is response to pathogenic and abiotic stress (Guo et al., 2010, Zhang et al., 2008). Similarly other highly conserved miRNA have been shown to be involved in multiple functions. For example miR393 is involved in pathogen defence, abiotic stress response, nitrate induced changes to root architecture and several aspects of development (Navarro et al., 2006, Godfroy et al., 2006, Messinese et al., 2007, Gleason et al., 2006, Schauser et al., 1999). In Arabidopsis, miR164 targets five NAC transcription factors
involved in vegetative and floral organ development (Laufs et al., 2004), regulates lateral root emergence in Zea mays (McNulty et al., 1996) and has also been linked to biotic and abiotic stress response (Kaló et al., 2005, Doyle, 1998).
1.10.2.1 miRNAs are regulators of auxin directed root formation
A frequent approach to miRNA discovery has been to employ transcriptome wide screening techniques to predict miRNAs and their targets (Chapter 3 of this thesis presents the results of of this same approach). These have shown that many highly conserved miRNA target
transcription factors involved in plant development (Jones-Rhoades and Bartel, 2004, Dai et al., 2011, Ding et al., 2011). Additionally these studies have shown members of the
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of miRNAs during root development. Auxin-miRNA feedback loops are also predicted to be highly represented given the observation that auxin response elements are frequently found in the promoter regions of auxin-associated miRNAs (Wall and Berry, 2008) (Pawlowski and Bisseling, 1996).
The importance of auxins in post-embryonic plant development has been long appreciated. It is not unexpected therefore that developmentally important miRNAs have also been linked to auxin regulation. Polar auxin transport produces localised influx/efflux events producing auxin gradients. The sites of auxin maxima then define regions of root organ primordia initiation. The auxin maxima induce stem cell niche formation and is therefore crucial to the determination of cell fate and correctly organised root architecture. The tight regulation of auxin signalling requires co-ordination between multiple genetic factors. Under low auxin conditions members of the Auxin/INDOLE3-ACETIC ACID (Aux/IAA) family repress members of ARF transcription factors (Tanabe and Nishibayashi, 2013). TRANSPORT INHIBITOR RESPONSE 1 (TIR1) and the closely related AUXIN F-BOX PROTEINs (AFBs) are induced by auxin in a dose dependent manner (Roy et al., 2002, Galloway et al., 2003). Binding of AUX/IAA by TIR or AFB proteins leads to ubiquitination and degradation by the 26S proteasome (Forster P et al., 2007).
Disruption of auxin at the transport, perception or response levels has been shown to have profound effects on root development (see (Liscum and Reed, 2002). During root
organogenesis, cytokinin acts as an antagonist to auxin function, restricting the induction the stem cell niche to a very small area thus restricting the size of the meristem (Dello Ioio et al., 2007).
In order to maintain the cells of the root meristem in a pluripotent state the QC must be prevented from differentiating. This requires control of auxin and cytokinin levels in a spatially precise manner. Auxin is obtained primarily through polar transport from the shoot and through local biosynthesis (Eamens et al., 2009, Blilou et al., 2005). Transport is directed by PINFORMED proteins leading to high concentrations of the auxin inducible transcription factor family PLETHORA (PLT). PLT 1-2 concentration radiates out from the QC defining various cellular states from QC maintenance, stem, cell identity, mitotic activity and
differentiation as dosage depletes moving away from the maxima (Galinha et al., 2007) . Concurrently a parallel pathway contributes to QC stem cell niche maintenance. The GRAS family transcription factor SHORTROOT (SHR). Expressed in the stele, SHR acts cell non-
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autonomously, via endosome trafficking, to activate another GRAS family transcription factor SCARECROW (Redmond et al.) in the QC, structural initials and endodermis (2009) (Gregg, 2011). Both SCR and SHR mutants result in loss of the QC (Duffin, 2010). SCR prevents cell division within the QC by repressing the cytokinin response regulator
ARABIDOPSIS RESPONSE REGULATOR 1 (ARR1) (Sabatini et al., 2003). Additionally, SHR/SCR in combination with down steam effectors regulate cell cycle genes controlling asymmetrical cell division and timing (Sabatini et al., 2003). The homeo-domain
transcription factor WUSCHEL-RELATED HOMEOBOX 5 (WOX5) is activated by SCR in the QC. WOX5 exerts influence on the distal region of the stem cell niche maintaining pluripotency and division of columella cells. WOX5 expression is also dependent on the AUXIN RESPONSE FACTORS ARF10 and ARF16. These, as well as ARF17, are targets of miR160 (Hill, 1999, Wang et al., 2005a).
When miR160 resistant versions of ARF16 and ARF17 are over-expressed in Arabidopsis pleotropic developmental defects are observed (Mallory et al., 2005, Lee et al., 1997, Wang et al., 2005b). Adventitious root formation also requires miR160/ARF17 interaction and is regulated by a feedback loop: ARF6 up-regulates miR160 and miR167 which are also down- regulated by ARF8 and ARF17 (Mallory et al., 2005, Van Breemen et al., 1984). ARF17 suppresses expression of three auxin-inducible Gretchen Hagen3 (GH3) genes reducing free auxin levels (Gutierrez et al., 2009, Zhou et al., 2014). In Arabidopsis auxin responsiveness is also regulated by miR390 which down-regulates ARF2, ARF3 and ARF4 via tasiRNA TAS3 and represses lateral root emergence (Boxman et al., 1998). miR390 is induced by auxin thereby forming part of an auxin–miR390-ARF auto-regulatory loop. In Arabidopsis and Zea mays miR164 is expressed in the pericycle during lateral root initiation where it is a negative regulator of root formation by limiting auxin signalling through the targeting of NAC1 (Guo et al., 2005, McNulty et al., 1996).
In M. truncatula, miR396 has been linked to meristem activity via repression of several transcription factor growth-regulating genes (GRFs) as well as two bHLH79. Over- expression of miR396 correlates with down-regulation of cell cycle genes and slows meristematic cell division (Trewavas, 2002).
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