Storage reserve mobilisation and seedling establishment in Arabidopsis.

During seed development large quantities of carbon, nitrogen and minor nutrients are stored from the mother plant. These will fuel post-germinative seedling establishment until chloroplast and root development are complete and photosynthesis can begin. Around 70% of our food comes from seeds, and much of the rest comes from animals fed on seeds, so seed storage reserves are of central importance to human existence (Bewley and Black, 1994). Carbon storage in the form of triacylglycerol (TAG) is a ubiquitous feature of seed plants, even in cereals that store the majority of their carbon as starch. During Arabidopsis seed development starch accumulates transiently but is eventually converted to TAG, which is stored in organelles known as liposomes, or oil bodies. Cells of the embryo and endosperm are packed full of oil bodies (Figure 1), which comprise up to 45% of the dry weight of the mature Arabidopsis seed (O'Neill et al., 2003). TAG accumulation depends on the action of genes that promote embryo identity and seed dormancy such as LEAFY COTYLE-DON1, FUSCA3 and ABSCISIC ACID INSENSITIVE 3, and requires the activity of the Apetala2 transcription factor WRINKLED1, which regulates carbon flow through glycolysis in the developing seed (Focks and Benning, 1998; Cernac and Benning, 2004). The close relationship of Arabidopsis to major and emerging oilseed crops makes the study of Arabidopsis fatty acid metabolism especially relevant, and the current state of the art knowledge gained from Arabidopsis underpins modern attempts to engineer plants to produce neutraceutical polyunsaturated fatty acids, to improve oil crops for biodiesel production, and for the provision of oil to replace dwindling and increasingly expensive petrochemical supplies (Thelen and Ohlrogge, 2002; see www.oilcrop.com). Figure 1. Transmission electron micrographs of imbibed Arabidopsis seeds showing embryo and endosperm cells packed with lipid or oil bodies. Abbreviations: LB, lipid bodies; Nu, nucleus; SPV, storage protein vacuole. This chapter will describe the pathways required for the breakdown and mobilisation of seed oil in Arabidopsis. This requires the hydrolysis of TAG by lipases and subsequent s-oxidation of the resultant fatty acids in the peroxisome. This produces acetyl-CoA, which is converted to citrate and then either used for respiration, or through the glyoxylate cycle and gluconeogenesis is converted into soluble sugars to support metabolism and growth (Figure 2). The activity of these pathways is tightly coordinated with the control of seed dormancy and germination. However, in Arabidopsis it has been shown that, in the final stages of seed maturation, seed oil content actually falls, indicating that reserve mobilisation begins prior to germination (Baud et al., 2002). This correlates with the appearance of transcripts for key genes in s-oxidation and the glyoxylate cycle (Schmid et al., 2005). Figure 2. An overview of the major metabolic pathways required for lipid reserve mobilization in germinating Arabidopsis seeds. 2.1 LIPOLYSIS Lipolysis is the first step of lipid reserve mobilisation, yet at present it remains the most enigmatic. Various lipases with activity against TAG have been purified from plants, including oilseed rape, maize, and castor bean, but no genetic evidence has been presented that any are required for TAG hydrolysis in germinating seeds. The well known acid lipase from castor bean endosperm has recently been cloned (Eastmond, 2004b). This lipase is anchored to oil bodies by a long hydrophobic stretch of amino acids and is highly active against the TAG triolein in vitro. Yet the acid pH optimum and peak of expression during seed development make this an unlikely candidate for the lipase required for post-germinative reserve mobilisation. Recently a candidate for Arabidopsis TAG lipase was identified by El-Kouhen et al., (2005). This protein shares weak homology with mammalian gastric lipases and was expressed during seed germination. However, the recombinant protein only showed high activity towards short chain TAGs not found in Arabidopsis seed storage reserves, and an insertion mutant still retained wild type TAG lipase activity and could break down TAG at the normal rate. A more likely candidate for Arabidopsis TAG lipase is the gene encoded by the sugar dependent 1 (sdp1) locus (Eastmond, 2006). The sdp1 mutant cannot break down seed storage lipid and shows poor hypocotyl elongation in the dark that can be promoted to wild type levels by applying an alternative carbon source such as sucrose. As we shall see, this phenotype is diagnostic for an impairment in storage reserve mobilisation (Eastmond et al., 2000b; Cornah et al., 2004; Penfield et al., 2004). The gene corresponding to this mutation has been cloned and the gene product possesses a patatin-like serine esterase domain also found in mammalian adipose tissue lipase and yeast TAG lipase (Zimmerman et al., 2004). SDP1 is active against long chain TAGs in vitro, and GFP fusions show that it is associated with oil bodies during germination. Hence SDP1 is the only candidate for plant TAG lipase for which convincing genetic evidence for function has been presented. SDP1 has only low activity against diacylglycerol and no activity against monoacylglycerol in vitro suggesting that further genes encoding these lipases are yet to be identified. The Arabidopsis genome contains one gene (At3g57140) closely related to SDP1, the corresponding protein showing 74% identity at the amino acid level. Expression of At3g57140 is detected in pollen and mature seeds according to Genevestigator. TAG oil bodies are also known to be associated with pollen and a novel group of oil-body associated proteins, termed oleosins have been shown to be present inside the pollen of Arabidopsis (Wu et al., 1997; Kim et al., 2002). It is thus tempting to speculate that this SDP1 homologue plays a role in hydrolyzing oil body TAGs in pollen.