Tony Millar

Research interests

Until recently it was thought that the majority of regulation was carried out by proteins, and that the DNA between protein-coding genes was nothing other than junk DNA. However this view has changed with the discovery of a new class of gene regulators called small RNAs. These molecules constitute an RNA-centric layer of control whose enormity has only become apparent in the last five years and may provide part of the regulatory requirements needed for the level of complexity observed in higher eukaryotes.

The lab focuses on one type of small RNA known as microRNAs (miRNAs), many of which have been shown to play pivotal roles in many developmental and physiological processes, in both plants and animals. MiRNA genes encode a transcript that forms an extensive secondary structure known as a stem-loop (Fig. 1A). This stem-loop is then processed into a small 21 nucleotide molecule (Fig. 1B) known as the ‘mature’ miRNA. This miRNA then guides the RNAi silencing machinery to complementary mRNA molecules, which results in the repression of those targeted mRNAs, hence silencing gene expression.

We have been using mutants in the model plant Arabidopsis, that fails to produce one type of miRNA known as miR159. This has dramatic consequences for the plant’s development, where the miRNA mutant develops curled leaves, smaller fruits and seeds (Fig 1). These traits are a consequence of the de-regulation of a family of genes that code for MYB transcription factors, where each family member has a sequence to which miR159 can bind to and then destroy the gene transcript (mRNAs). This regulation is shown with a reporter gene (gives a blue colour) that has been joined to a MYB gene; in the miRNA mutant there is strong activity (indicating loss of regulation), whereas in the normal wild-type plant no activity can be detected, indicating the gene is being naturally 'silenced'. Correlating this gene activity with the mutant traits demonstrates that this natural gene silencing mechanism is critical for proper plant development. Understanding this recently discovered form of gene regulation may provide insights on how to manipulate leaf shape, fruit and seed size, all extremely important agronomic traits.

Figure of genetic variants of Arabidopsis

Figure 1

Recent research highlights MiR159 has a narrow functional specificity To define miRNA targets, the commonly adopted approaches of bioinformatic and molecular analysis has predicted that more than twenty target genes for the Arabidopsis miR159 family. However using genetic analysis we determined that the functional specificity of the major miR159 family members, miR159a and miR159b is limited to only two targets, MYB33 and MYB65. This was shown by creating a mir159ab/myb33/myb65 quadruple mutant, that suppresses all the mir159ab pleiotropic developmental defects (Fig 2). This narrow functional specificity is similar to what has been found in animal miRNA systems, where the loss-of-function phenotypes found in the miRNAs lin-4 and let-7, can be suppressed by mutating a single target gene, despite these miRNAs being predicted to target many genes. Hence, only a few physiologically relevant targets from a much broader set of predicted targets is an emergent and unifying theme in plant and animal miRNA biology. Allen et al., 2007, Proceedings of the National Academy of Science USA 104:16371-6.

Rosette phenotypes of the various genotypes

Figure 2. Rosette phenotypes of the various genotypes

The dynamic nature of miRNA: target relationships. The above finding raised the question as to what is the selective pressure that drives the maintenance of conserved miR159 binding sites in other target genes? Examination of the third miR159 family member, miR159c, found that it was quiescent in nature, being very lowly expressed, but had an overlapping transcriptional domain with many of the other conserved miR159 targets, none of which appeared to be miR159 regulated as determined by functional analysis. Therefore this miR159c:target gene regulatory module in anthers appears inert or defunct, highlighting that even conserved miRNAs are likely to be undergoing dynamic changes leading to different regulatory outcomes. This study also highlight some of the limitations of the currently used methodologies for determining functionally significant miRNA regulation Allen et al., 2010 Silence 1:18.

Figure showing examples of Arabidopsis mutations

MiR159 controls Programmed Cell Death (PCD).To help elucidate the biological role of this miRNA pathway, we performed micro-array analysis on mir159ab (high MYB33 and MYB65 expression) and wild-type plants. Over 100 genes were up-regulated in mir159ab (P <0.005, 2-fold increase), and expression of these genes were correlated with inhibition of growth. Many of these, including the most up-regulated, are indicative of program cell death (PCD). Consistent with this, MYB33/MYB65 protein being found almost exclusively in the aleurone in seeds and tapetem in anthers, cell layers that undergo PCD. Functional analyse showed that PCD processes are inhibited in these tissues in mutant myb plants. In vegetative tissues of mir159ab (high MYB33 and MYB65) growth was inhibited (Figure 4). Therefore these “GAMYB-like” genes, which were thought to promote growth and flowering in fact inhibit growth and promote PCD. Alonso-Peral et al., 2010 Plant Physiology 757-771.Figure showing examples of Wildtype and mutant Arabidopsis

Some of our recent publications have appeared on the cover

journal covers

Recent grants

ARC Discovery grants

  • 2013-2015: Use of Molecular sponges to inhibit small RNA activity in plants - $490 K
  • 2011-2013: Plant microRNA systems: investigating slicing versus translational repression and the development of an anti viral defense mechanism - $270 K
  • 2006-2009: MicroRNA analysis of gene expression and development - $312 K

Major equipment grant

  • 2008 - Plant imaging equipment - $125 K.

Selected publications

Wong G, Millar AA. (2022) TRUEE; a bioinformatic pipeline to define the functional miRNA targetome of Arabidopsis. The Plant Journal. 110, 1476-1492. doi: 10.1111/tpj.15751. Epub ahead of print. PMID: 35352405.

Wang N, Millar AA (2021) Use of mRNA-Interactome Capture for Generating Novel Insights into Plant RNA Biology. In G Tang et al., (eds), RNA-based Technologies For Functional Genomics in Plants DOI 10.1007/978-3-030-64994-4_5

Luo J, Butardo VM Jr, Yang Q, Konik-Rose C, Colgrave ML, Millar A, Jobling SA, Li Z. (2020) The impact of the indica rice SSIIa allele on the apparent high amylose starch from rice grain with downregulated japonica SBEIIb. Theoretical and Applied Genetics. doi: 10.1007/s00122-020-03649-2.

Zheng Z, Wang N, Jalajakumari MB, Blackman L, Shen E, Verma S, Wang MB, Millar AA (2020) miR159 represses a constitutive pathogen defense response in tobacco. Plant Physiology 182, 2182-219 doi: 10.1104/pp.19.00786

Millar AA (2020) The Function of miRNAs in Plants. Plants (Basel)

Asadi Khanouki M, Rezanejad F, Millar AA (2019) Sequence and functional analysis of a TERMINAL FLOWER 1 homolog from Brassica juncea: a putative biotechnological tool for flowering time adjustment. GM Crops & Food doi: 10.1080/21645698.2011707340

Millar AA, Lohe A, Wong G (2019) Biology and function of miR159. Plants (Basel) 8, 255. doi:3390/plants8080255

Wong G, Millar AA (2019) The use of microRNA decoy technologies to inhibit miRNA function in Arabidopsis. In, S de Folter (ed) Plant microRNAs: Methods and Protocols, Methods of Molecular Biology vol. 1932, Springer Science, New York, pp 227-238.

Schumann U, Lee JM, Smith NA, Zhong C, Zhu JK, Dennis ES, Millar AA, Wang MB (2019) DEMETER plays a role in DNA demethylation and disease response in somatic tissues of Arabidopsis. Epigenetics 14, 1074–1087. doi:10.1080/15592294.2019.1631113.

Wong G, Alonso-Peral M, Li B, Li J, Millar AA (2018) MicroRNA MIMIC binding sites: minor flanking nucleotide alterations can strongly impact MIMIC silencing efficacy in Arabidopsis. Plant Direct 2, e00088.

Crisp PA, Smith AB, Ganguly DR, Murray KD, Eichten SR, Millar AA, and Pogson BJ (2018) Pol II read-through promotes expression of neighbouring genes in SAL1-PAP-XRN retrograde signaling. Plant Physiology 178, 1614-1630.

Medina C, da Rocha M, Magliano M, Ratpopoulo A, Revel B, Marteu N, Magnone V, Lebrigand K, Cabrera J, Barcala M, Silva AC, Millar A, Escobar C, Abad P, Favery B and Jaubert-Possamai S (2017) Characterization of microRNAs from Arabidopsis galls highlights a role for miR159 in the plant response to the root-knot nematode Meloidogyne incognita. New Phytologist 216, 882-896.

Zheng Z, Reichel M, Deveson I, Wong G, Li J and Millar AA (2017) Target RNA secondary structure is a major determinant of miR159 efficacy. Plant Physiology 174, 1764-1778.

Reichel M, Liao Y, Rettel M, Ragan C, Evers M, Alleaume A-M, Horos R, Hentze MW, Preiss T and Millar AA (2016) In planta determination of the mRNA-binding proteome of Arabidopsis etiolated seedlings. The Plant Cell 28, 2435-2452.

Li Y, Alonso-Peral M, Wong G, Wang M-B and Millar AA (2016) Ubiquitous miR159 repression of MYB33/65 in Arabidopsis rosettes is robust and is not perturbed by a wide range of stresses. BMC Plant Biology 16, 179.

Luo J, Ahmed R, Kosar-Hashemi B, Larroque O, Butardo VM Jr, Tanner GJ, Colgrave ML, Upadhyaya NM, Tetlow IJ, Emes MJ, Millar AA, Jobling SA, Morell MK, Li Z. (2015). The different effects of starch synthase IIa mutations or variation on endosperm amylose content of barley, wheat and rice are determined by the distribution of starch synthase I and starch branching enzyme IIb between the starch granule and amyloplast stroma. Theoretical and Applied Genetics 128, 1407-1419.

Reichel M and Millar AA (2015) Specificity of plant microRNA target MIMICs: Cross-targeting of miR159 and miR319. Journal of Plant Physiology 180, 45-48.

Luo J, Jobling SA, Millar AA, Morell MK and Zhongyi Li (2015) Allelic effects on starch structure and properties of six starch biosynthetic genes in a rice recombinant inbred line population. Rice 8, 15.

Reichel M, Li Y, Li J and Millar AA (2015) Inhibiting plant microRNA activity: molecular SPONGEs, target MIMICs and STTMs all display variable efficacies against target microRNAs. Plant Biotechnology Journal 13, 915-926.

Li J, Reichel M, Li Y and Millar AA (2014) The functional scope of plant microRNA-mediated silencing. Trends in Plant Science 19, 750-756.

Li, J, Reichel, M and Millar AA (2014) Determinants beyond both complementarity and cleavage govern miR159 efficacy in Arabidopsis. PLoS Genetics 10, e1004232.

Deveson I, Li J and Millar AA (2013) MicroRNAs with analogous target complementarities perform with highly variable efficacies in Arabidopsis. FEBS Letters 587, 3703-3708.

Allen RS, Nakasugi K, Doran R, Millar AA and Waterhouse PM (2013) Facile mutant identification via a single parental backcross method and application of whole genome sequencing based mapping pipelines. Frontiers in Plant Science 4, 362.

Deveson I, Li J and Millar AA (2013) Expression of human ARGONAUTE 2 inhibits endogenous microRNA activity in Arabidopsis. Frontiers in Plant Science 4, 96.

Li J and Millar AA (2013) Expression of a microRNA-resistant target transgene misrepresents the functional significance of the endogenous microRNA:target gene relationship. Molecular Plant 6, 577-580.

Alonso-Peral MM, Sun C and Millar AA (2012) MircoRNA159 can act as a switch or tuning microRNA independently of its abundance in Arabidopsis. PLoS ONE 7, e34751.

Agius C, Eamens AL, Millar AA, Watson JM and Wang M-B (2012) RNA silencing and antiviral defense in plants. In, JM Watson and M-B Wang (eds) Antiviral resistance in Plants: Methods and Protocols, Methods of Molecular Biology vol. 894, Springer Science, New York, pp 17-38.

Fahim M, Millar AA, Wood CC and Larkin PJ (2012) Resistance to Wheat streak mosaic virus generated by expression of an artificial polycistronic microRNA in wheat. Plant Biotechnology Journal. 10, 150-163.

Allen RS and Millar AA (2012) Genetic and molecular approaches to assess microRNA function. In, R. Sunkar (ed.), MicroRNAs in Plant Development and Stress Responses, Signaling and Communication in Plants 15, Springer-Verlag Berlin Heidelberg, pp 123-148.

Reichel M, Li J and Millar AA (2011) Silencing the silencer: strategies to inhibit microRNA activity. Biotechnology Letters 33, 1285-92.

Greenup AG, Sasani S, Oliver SN, Walford SA, Millar AA and Trevaskis B (2011) Transcriptome analysis of the vernalization response in barley (Hordeum vulgare) seedlings. PLoS One 6, e17900.

Allen, RS, Li J, Alonso-Peral MM, White RG, Gubler F and Millar AA (2010) MicroR159 regulation of most conserved targets in Arabidopsis has negligible phenotypic effects. Silence 1, 18

Alonso-Peral MM, Li J, Li Y, Allen RS, Schnippenkoetter W, Ohms S, White RG, Millar AA (2010) The microR159 regulated GAMYB-like genes inhibit growth and promote programmed cell death in Arabidopsis. Plant Physiology 154, 757-771.

Fahim M, Ayala-Navarrete L, Millar AA, Larkin PJ (2010) Hairpin RNA derived from viral NIa gene confers immunity to wheat streak mosaic virus infection in transgenic wheat plants. Plant Biotechnology Journal 8, 821-834.

Agius, C, Eamens, AL, Millar AA, Wang, M-B (2010) Regulatory giants join forces. Frontiers in Biology, 5, 5-7

Barrero JM*, Millar AA*, Griffiths J, Czechowski T, Scheible WR, Udvardi M, Reid JB, Ross JJ, Jacobsen JV, Gubler F (2010) Gene expression profiling identifies two regulatory genes controlling dormancy and ABA sensitivity in Arabidopsis seeds. The Plant Journal 61, 611-622 * = equal first author

Cazzonelli CI, Millar T, Finnegan EJ, Pogson BJ (2009) Promoting gene expression in plants by permissive histone lysine methylation. Plant Signaling and Behavior 4, 484-488.


Allen, RS, Li J, Stahle MI, Dubroué A, Gubler F, Millar AA (2007) Genetic analysis demonstrates functional redundancy and the major target genes of the Arabidopsis miR159 family. Proceedings of the National Academy of Sciences, USA 104, 16371-16376. 

Millar AA, Jacobsen JV, Ross JJ, Helliwell CA, Poole AT, Scofield G, Reid JB, Gubler F. (2006) Seed dormancy and ABA metabolism in Arabidopsis and barley: the role of ABA 8’ hydroxylase. The Plant Journal 45, 942-954.

Millar AA, Waterhouse PM (2005) Plant and animal microRNAs: similarities and differences. Functional and Integrative Genomics 5, 129-135.

Gubler F, Millar AA, Jacobsen JV (2005) Dormancy release, ABA and pre-harvest sprouting. Current Opinion in Plant Biology 8, 183-187.


Millar AA, Gubler F (2005) The Arabidopsis GAMYB-like genes, MYB33 and MYB65, are microRNA-regulated genes that redundantly facilitate anther development. The Plant Cell 17, 705-721.

All publications

  • Convenor; Masters of Biotechnology program
  • Convenor; BIOL8702, Advanced Molecular Biology Techniques
  • Co-convenor; BIOL2162 & BIOL6162, Molecular Gene Technology.