Biochemical analyses of AtDRM1 and AtDRM – Project topics materials

Get Complete Project Material File(s) Now! »

The DRM1/ARP protein family

Database searches show that the DRM1/ARP family is unique to plants, with homologues found in both monocotyledons and dicotyledons (Park and Han, 2003). Multiple protein sequence alignments show high conservation of the DRM1/ARP protein product, with two domains clearly identifiable at the amino-terminus and at the carboxy-terminus (Park and Han, 2003; Wood et al., 2013). This high conservation across species, and indeed monocotyledons and dicotyledons, suggests a potential importance of the action of this protein in the normal development and physiology of plants. Moreover, the protein product is conserved in its size, with all eleven species
analysed by Park and Han (2003) between 11 and 14 kDa (between 109 and 126 amino acid residues). GFP fusion constructs of EuNOD-ARP1 (EuNOD-ARP1; Elaeagnus umbellata nodule AUXIN-REPRESSED PROTEIN GENE-1), a DRM1/ARP homologue in the ornamental shrub E. umbellata (Japanese silverberry, Autumn-Olive), as well as GFP fusion constructs of Brassica rapa DRM1 and ARP1, suggest that it is cytosollocalised (Kim et al., 2007; Lee et al., 2013). However, in silico prediction shows AtDRM1 in the nucleus and mitochondria, possibly suggesting differing roles for family members from different species. No characterised localisation signals or DNAbinding domains are found in the protein sequence. Additionally, the PFAM motif (PF05564), ‘dormancy/auxin associated protein’, represents almost the entire protein, leaving few hints on the role of this protein in planta.
Recently evidence has emerged for DRM1/ARP family members as intrinsically disordered proteins (IDPs) (Wood et al., 2013). IDPs, found in plants and animals, have a large proportion of their sequence yielding no fixed tertiary structure, yet still providing some biological function (Dunker et al., 2001). Signal transduction and transcriptional regulation are key processes within which structural disorder is prominent (Iakoucheva et al., 2002; Minezaki et al., 2006b). A well characterised example of IDPs in the plant kingdom is the late embryogenesis abundant (LEA) class of proteins, involved in abiotic stress tolerance (Garay-Arroyo et al., 2000; Mouillon et al., 2006) as chaperones for other stress response genes (Chakrabortee et al., 2007; Kovacs et al., 2008a; Kovacs et al., 2008b). ERD10 and ERD14 (EARLY RESPONSE TO DEHYDRATION) are members of the dehydrin type of LEA proteins, shown to be both intrinsically disordered and responding to high salinity, drought and low temperature stresses in Arabidopsis (Kovacs et al., 2008b). Similarly, a (LEA)-like phosphoprotein (CDeT11-24) whose expression is associated with periods of dessication, has been identified in the resurrection plant Craterostigma plantagineum (Petersen et al., 2012). Indeed with these findings in mind, DRM1/ARP family members may have roles in chaperoning proteins critical to the no-growth phase in tissues.
Despite the high degree of conservation across species, the biological significance of the DRM1/ARP protein family remains unknown.
Potential role of DRM1/ARP in the growth/no-growth decision outside of meristematic tissues Despite being a commonly used marker for dormancy, DRM1/ARP is expressed in tissues other than dormant axillary buds with high expression often localised to nongrowing tissues.
Fruit development is an example of the growth/no-growth homeostasis in floral tissues (cf. vegetative tissues) and as such provides further evidence of DRM1/ARP’s association with non-growing tissues. For example, one DRM1/ARP homologue, AP1, was first discovered in cortical tissue from ripe apple fruit (Lee et al., 1993). In addition, Reddy and Poovaiah (1990) showed a positive correlation between strawberry (Fragaria ananassa) fruit growth and repression of a homologue identified from a library of fruit starved for auxin, λSAR5, while being highly expressed in mature fruit which have stopped growing. More recently, Pyrus pyrifolia ARP1 (PpARP1) and PpARP2 expression was detected in the mesocarp tissue of Pear (Pyrus pyrifolia) fruit (Shi et al., 2013).
Similar data has been seen in kiwifruit with AdDRM1 levels reaching a peak in ripe, mature fruit (unpublished: M. Wood) and in tomato where high levels of the gene transcript were localised to the ovaries, with a decrease exhibited after fruit set (Vriezen et al., 2008). It is hypothesised that unpollinated ovaries are in a temporally dormant state, supported by expression of cell cycle genes induced after fruit set (Vriezen et al., 2008). As these tissues may have included seeds, and in silico expression profiles show seeds have high levels of SlDRM1 expression, further analysis is required in these species to clarify whether this increase in expression is a result of increases in the maturing fruit flesh, or rather in the associated maturing seeds.
Increased DRM1/ARP gene expression has also been described in tobacco flowers (Steiner et al., 2003); mature stems, leaves and roots of pea (Stafstrom et al., 1998); dehisced pollen of tobacco (Steiner et al., 2003); and root nodules of E. umbellata (Kim et al., 2007). While not at particular high levels, DRM1ARP transcript has also been detected in petioles, seeds, various floral organs, and across trunk wood of mature trees (Lee et al., 1993; Park and Han, 2003; Ross et al., 1992; Stafstrom et al., 1998; Steiner et al., 2003).
A classical example of tissue aging and transitioning into growth suppression is provided in the leaf senescence process. Arabidopsis max2 mutants, already described earlier for their hyper-branching phenotype, also exhibit increased longevity during leaf senescence (Woo et al., 2001). In Arabidopsis, MAX2 transcript expression peaks during the early stages of senescence; while AtDRM1 expression continually increases throughout senescence, reaching a maximum when yellowing begins, at which point expression remained reasonably constant (Breeze et al., 2011). These data suggest that DRM1/ARP is involved in leaf senescence, but later in the process than MAX2, raising the question; is MAX2 controlling DRM1/ARP?
An interesting hypothesis given by Park and Han (2003) is for a putative in planta role for DRM1/ARP family members in repression of cell elongation. The authors introduced an inverse correlation between expression of the DRM1/ARP homologue in black locust, RpARP (Robinia pseudoacacia auxin repressed protein) and the elongation zone of 7-day old hypocotyls. Possible contradictory evidence is found in the hyperelongated phyb mutant which has increased DRM1 transcript expression in Arabidopsis axillary buds (Finlayson et al., 2010). To clarify this, AtDRM1 transcript expression would need to be measured in the hyper-elongating hypocotyl tissue of the phyb mutant.

READ Cytokines and intracellular signalling domains

1 Introduction
1.1 Dormancy
1.2 The dormancy marker DRM1/ARP: associated with dormancy but a broader role in planta
1.3 Aims of this thesis
2 AtDRM1/ARP family members
2.1 Introduction
2.2 Methods .
2.3 Results .
2.4 Discussion
2.5 Conclusions .
3 Regulation of AtDRM1 and AtDRM2 .
3.1 Introduction
3.2 Methods
3.3 Results
3.4 Discussion .
3.5 Conclusions .
4 Down-regulation of AtDRM1 and AtDRM2
4.1 Introduction .
4.2 Aims of this chapter
4.3 Methods
4.4 Results .
4.5 Discussion
4.6 Conclusion
5 Biochemical analyses of AtDRM1 and AtDRM
5.1 Introduction .
5.2 Methods
5.3 Results .
5.4 Discussion .
5.5 Conclusion
6 Discussion and Conclusions
6.1 Moving on from historical themes in DRM1/ARP research
6.2 Complexity of the DRM1/ARP family
6.3 AtDRM1 and AtDRM2 in temporary growth arrest outside of dormancy
6.4 A role for AtDRM1 and AtDRM2 in protective stress responses
6.5 Challenges in studying AtDRM1/ARP .
6.6 Proposed model for the role of AtDRM1 and AtDRM2 in plants
6.7 Future work
6.8 Conclusions and significance
7 Appendices
References