Fases de posicionamiento (Codina, 2004).

8.2.1 siRNA are the Predominant Class of smRNA

Maize wild-type (WT) datasets showed an abundance of 24nt siRNA, approximately 7-fold higher than the next most abundant smRNA. Equally, the diversity of 24nt was higher than other smRNA; the population of 24nt smRNA was composed of more unique smRNA than

other classes (Figure 5.3). Similarly in other plant models and existing maize data, siRNA form the majority of smRNA. There are fluctuations in the distribution of smRNA classes between tissues, where some tissues express more miRNA, for example. In the rapidly dividing meristematic area (MA) sampled in this work, the dominance of siRNA suggests a link to RdDM and that TGS is an important mechanism of gene regulation.

In RdDM compromised plant systems, the abundance of siRNA is reduced; typically, such plants are unable to produce siRNA due to a lack of Pol IV, RNA-DEPENDENT RNA POLY- MERASE2 (RDR2) orDCL3homologues. The maize homologue ofRDR2,Mop1, is unable to convert Pol IV transcripts into dsRNA prior to digestion into smRNA. In agreement with Nobuta et al. (2008), ourmop1/mop1datasets showed a reduced capacity to produce, but not lack of, siRNA and concomitant proportional increase in miRNA and 22nt smRNA. Our datasets further support the observation that 22nt smRNA are unaffected by the lack of MOP1 (Supplementary Figure C1). Previous characterisation of the smRNA population in maize used immature ears whereasmop1-1datasets presented here utilise MA showing thatMop1 is an important tissue-independent protein in the RdDM pathway.

8.2.2 miRNA Provide an Immediate Stress Response

miRNA regulate gene expression by PTGS. Through complementarity to a gene transcript, miRNA direct enzymatic cleavage of messenger RNA (mRNA) thereby limiting translation (Carthew and Sontheimer 2009, Mallory and Vaucheret 2006). In this analysis, smRNA datasets were compared to known miRNA using BLAST and the response of miRNA families to temperature stress tested (Section 5.3). Figure 5.6 shows the over-representation of thymine at the 5’ end of smRNA with a significant BLAST alignment. The 5’ terminal nucleotide is required to ensure smRNA are bound to the correct AGO. In AGO pull-down experiments, smRNA bound to AGO1 have exhibited enrichment for a 5’ terminal thymine (Jeong et

al. 2013, Mi et al. 2008). The datasets presented here do not account for smRNA-AGO interactions but it is reassuring that the smRNA identified as miRNA derived show an expected characteristic. We can be confident, therefore, that smRNA identified as miRNA provide a reasonable prediction of miRNA expression.

In response to temperature stress, three-quarters of detected miRNA families showed environment-dependent expression. The response of miRNA was similar to both environ- mental stresses at the early time point but diverged following recovery where misregulation was maintained during recovery for a higher proportion of heat affected miRNA (Section 5.3). These results indicate that environmental stress causes similar miRNA responses but that the response is attenuated to stress severity and that heat stress is more disruptive than cold to maize. Few miRNA families were identified that have not been linked to environmental stress but some were identified that are linked to development. The maize stress response may therefore be more closely linked to development than in other model plants but this could also indicate that developmental stage and tissue type have an impact on the stress response. Our datasets reaffirm that miRNA are an important part of the stress response and provide evidence that their expression can be maintained during recovery.

Cold environmental stress affected more miRNA families than heat, but heat-affected miRNA were more frequently maintained, and for both stresses very few were affected only after recovery (Table 5.3). This highlights a similarity in the miRNA-mediated stress response; that miRNA confer immediate adaptations to stress and suggested that miRNA were not regulated by epigenetic mechanisms, unlike protein-coding genes where such mechanisms may be responsible for delayed gene expression responses.

More miRNA families were misregulated inmop1-1datasets than environmentally stressed WT datasets. These differences may be due to the lack of siRNA providing a more accurate measure of miRNA expression in mop1/mop1 datasets, where they were 10-fold more

abundant. The comparatively low frequency of miRNA derived smRNA in WT datasets may have led to an underestimate of stress-responsive miRNA families due to increased noise. However, miRNA were not the focus of this project and further work to characterise miRNA expression during recovery from environmental stress could use an AGO enrichment approach to provide more accurate measurement of miRNA expression.

8.2.3 siRNA Reduction at Transposable Elements

The RdDM pathway is a key mechanism in the regulation of transposable elements; RdDM deficient plants suffer increased rates of transposition and altered sensitivity to stress. How- ever, there are different types of transposable elements and the RdDM pathway does not regulate them all similarly.

Transposable elements are found throughout the maize genome and a high proportion of the genome has similarity to known transposable elements. The smRNA datasets described here showed that the interaction of smRNA to the highly repetitive retrotransposons was different to the interaction with DNA transposons (Section 5.2). In response to environmental stress, the abundance of siRNA targeting flanking regions reduced at retrotransposons which may have led to increased transposon expression. DNA transposons were targeted across the entire element and their genome-wide smRNA profiles were not as affected by environmental stress.

Thousands of transposable element insertions were identified as having potentially altered RdDM activity due to environmental stress, including DNA transposons (Section 5.5). Highly repetitive retrotransposons were also identified as stress-responsive by these datasets. The retrotransposon class of transposable elements increase genome size when they transpose and produce genetic diversity in response to stress (Bucher et al. 2012). Transposable