Seed germination: a transition from embryonic to vegetative development

The end of the maturation phase represents the transition from an embryonic to a vegetative state. To allow germination and seedling development to occur, maturation phase gene expression must come to an end. Repression of maturation-specific gene expression involves two mechanisms; factors that directly repress specific maturation genes and factors that repress the key LEC and ABI3 regulatory proteins (Bouyer et al., 2011; Verdier and Thompson, 2008). Both mechanisms typically achieve seed gene repression through the modification of chromatin structure however these mechanisms are not well understood (Zhang and Ogas, 2009; Tang et al., 2008; Gutierrez et al., 2007). Several factors have been identified that repress seed maturation genes by covalent and non-covalent modification of histone proteins and DNA [HISTONE DEACETYLASE6 and HISTONE DEACETYLASE19 (Tanaka et al., 2008), FERTILIZATION INDEPENDENT ENDOSPERM (Bouyer et al., 2011), PICKLE (Aichinger et al., 2009; Zhang et al., 2008) and BRAHMA (Tang et al., 2008)]. Seed gene repression can also be achieved through transcriptional repression by ARABIDOPSIS 6B-INTERACTING PROTEIN 1-LIKE 1 (ASIL1) (Gao et al., 2009) and VP1/ABI3-LIKE1 (VAL1) and VAL2 (Suzuki and McCarty, 2008; Suzuki et al., 2007; Tsukagoshi et al., 2007).

1.4.2 Mobilization of storage reserves

Once the seed receives sufficient cues for germination, the embryo resumes growth. During early seedling development, storage reserves are used to promote seedling establishment until chloroplast and root development are complete and photosynthesis can begin (Penfield et al., 2008). The seed imbibes water, cells expand and the seed coat ruptures. Radicle cells divide and elongate causing the radicle to emerge from the seed coat. The radicle continues to elongate into the substrate and root hairs form shortly afterwards. Subsequently, cotyledons emerge from the seed coat, the hypocotyl elongates and positions the unfolding cotyledons over the seed coat. The cotyledons expand and

they begin to green. At the subcellular level, storage vacuoles transition to lytic vacuoles (LVs) as proteins are mobilized. At the same time, oil bodies are degraded. Concomitant with cotyledon greening, proplastids multiply and differentiate to give rise to chloroplasts (Mansfield and Briarty, 1996; Huang, 1992).

At early germination, the embryo is not yet equipped to be self-sufficient so it relies heavily on heterotrophic growth. Lipid and protein reserves are first metabolized in the layer of endosperm cells. Later, seedling reserves are first mobilized in the radicle and hypocotyl (Kaneko and Keegstra, 1996; Mansfield and Briarty, 1996). During mobilization of storage reserves, many hydrolytic enzymes are required to degrade storage reserves. Proteases hydrolyse SSPs to amino acids, which become incorporated into newly synthesized proteins. Phytate, a storage form of phosphorus and minerals in the storage vacuoles, is hydrolysed by phytase and the solubilized minerals are released. Triglycerides are hydrolysed by lipases and the fatty acids are used to produce sugars and ATP (Bethke et al., 1998). As reserves are depleted from the cotyledons to support embryo growth, the cotyledons are transformed into photosynthetic organs which will eventually allow the seedling to convert to autotrophic growth. The transition of cotyledons from a storage to a photosynthetic tissue occurs 48-60 hr after imbibition (Mansfield and Briarty, 1996).

1.4.3 Storage protein reserves are accumulated in leaves

Vegetative tissues transiently accumulate storage proteins in vacuoles to serve as a supply of nutrients for growth and development. When nutrients are plentiful, plants assimilate them into vegetative storage proteins (VSPs) to build up a reserve of nutrients. These proteins are an important source of carbon, sulfur and especially nitrogen. When required by the plant, VSPs are degraded to release amino acids that will be redistributed for other metabolic purposes (Rennenberg et al., 2010; Staswick, 1994).

All proteins sequester amino acids which are later released as proteins turn over. Thus, all plant proteins can be considered as storage reserves because they can provide a

nutritional need. But what sets VSPs apart from other proteins? Unlike VSPs, most proteins are not abundantly accumulated in vegetative tissues (Staswick, 1994). An exception is Rubisco, the most abundant protein on Earth. Rubisco can contribute up to 50% of the soluble leaf protein and 20-30% of the total leaf nitrogen (Feller et al., 2008). Many consider Rubisco to be a storage protein but others argue that it is not a true VSP because it is required for other functions rather than storage. To be specific, the biosynthesis and degradation of Rubisco are regulated according to the need for its metabolic function and not for storage purposes (Rennenberg et al., 2010; Staswick, 1994). However many proteins that are classified as VSPs have alternate roles. The VSPs in soybean leaves are acid phosphatases (Berger et al., 1995), sweet potato tuber sporamin is a trypsin inhibitor (Yeh et al., 1997) and potato tuber patatin is a lipid hydrolase (Andrews et al., 1988).

VSPs were first described in soybeans and are well-characterized in this species (Wittenbach, 1983). Two soybean VSPs were discovered, VSPα and VSPβ. They are glycosylated polypeptides that are ~ 80% identical and have no sequence similarity with known SSPs. These proteins exist as homo- and heterodimers and accumulate in vacuoles of cells associated with the vascular system in stems, leaves, flowers and pods. Soybean VSPs can accumulate to as much as 50% of the total leaf protein but levels can decline to 1%. Their expression is enhanced by wounding, high nitrogen nutrition, drought stress, and the plant growth regulator jamonic acid (Utsugi et al., 1998; Berger, 1995; Staswick, 1994). Two genes homologous to soybean VSPs were identified in Arabidopsis (Berger et al., 1995). Arabidopsis VSPs are induced by similar stimuli and act as acid phosphatases but they primarily accumulate in flowers. However Liu et al. (2005) raised the question of whether they should be classified as VSPs because they have not been demonstrated to function as storage proteins. Thus, VSPs are recognized to exist in many plant species but identification and characterization of these proteins seems to be made difficult because many have alternate functions.

1.5 A portrayal of three prominent organelles involved in embryonic and vegetative