We began dissecting the process of turion formation in duckweeds. Usually turion development occurs in late summer or early autumn because of starvation and lower temperatures . Spirodela turions can also be induced under controlled laboratory conditions by increasing the concentration of ABA in the growth medium [11, 13], decreasing temperature , or depriving phosphorus in the medium . Here, we have taken advantage of ABA as an inducer and could reproduce the morphological changes that occur during turion formation. Turions are germinated into new fronds in the presence of light and nitrogen in the following spring using starch storage as an energy source [26, 27]. Therefore, the drastic starch accumulation during turion formation marks a turning point in the switch process from low-starch fronds to high-starch turions.
The reported contents of starch varied from 14% to 43% depending on the species, developmental states (fronds, resting fronds, or turions)  and tested methods [5, 29]. Starch content could even go up to 75% of the dry weight in resting fronds of Spirodela oligorrhiza (renamed into Landoltia punctata) growing in phosphor-deficient cultures , a level that is comparable to cereal seeds of corn, sorghum and wheat . Even though regular fronds have as low as 16% starch in dry mass, turions of S. polyrhiza can reach up to 62% starch . Our use of exogenous ABA produces the same developmental switch, as the different morphological features are easily distinguishable. The switch is rapid, providing advantages for biochemical and physiological analysis . We obtained 60.1% starch from dry mass after 2 weeks of ABA induction (Figure 2a), which is comparable to the Henssen's study. The size of mature starch grains from turions was around 4 μm in diameter as estimated by TEM (Figure 3e and 3f), whereas starch grains from wheat, corn and rice reach a size of 30 μm, 25 μm and 20 μm, respectively . In a different study, the size of starch granules illuminated by red light for different times have also been measured using SEM scans arriving at similar values . Interestingly, it has been suggested that smaller starch granules are more easily hydrolyzed into sugars than larger ones, regardless of botanical source . After 72 h of continuous irradiation, the sizes of starch granules in turions are significantly reduced to about 1.5 μm . Although duckweeds might have adapted to rapidly switch back to a growth phase faster than seed plants, this property also might provide a more efficient way for producing bio-ethanol than from maize.
Amyloplasts in non-photosynthetic tissue, such as seeds, roots, and stems, which lack chlorophyll and internal membranes, are the main organelles responsible for the synthesis and storage of starch granules in most plants. However, turions remain green or dark-green throughout their development (Figure 1b and 4b). The plastids in turions, where starch synthesis takes place, still retain abundant stacks of thylakoids (Figure 3e and 3f). It therefore suggests that chloroplasts with a simple structure as in duckweeds can function both as source and sink. The starch-storing plastids of turions are directly derived from chloroplasts, and retain chloroplast-like characteristics throughout their development. This adaptation greatly saves energy by directly depositing sucrose generated from photosynthesis into starch storage without the need for transport through a vascular system and the use of a glucose phosphate transporter . A similar system exists also in a non-aquatic plants such as pea embryos, where starch-storing plastids also directly originate from chloroplast [34, 35]. Moreover, using TEM light-induced degradation of starch granules in turions of Spirodela polyrhiza also exhibited a transition from amyloplasts to chloroplasts . Both studies would demonstrate that differentiation from chloroplast to amyloplast could be reversed based on physiological changes. Indeed, the cell structure of turions appears to be well organized for its function. Its lack of intercellular air space and presence of smaller vacuoles allow them to survive in deep water, where the temperature is more moderate than on the surface. The numerous starch grains provide a bank of energy when turions germinate in the following spring. This life cycle is also consistent with starch content in fronds and turions.
Because starch biosynthesis is an important feature for the developmental switch from fronds to turions, it also provides us with the first entry point to dissect the developmental regulation of turion formation. Therefore, we reasoned that the first step in this line of investigation consists of the identification and characterization of key regulatory genes known in starch biosynthesis, which are the ADP-glucose pyrophosphorylases. We successfully cloned three copies of APLs of Spirodela polyrhiza. APLs are expressed in different organs of grass species, type 1 in leaves, type 2 and type 3 in seeds, and type 4 in both seeds and leaves [21, 36]. Based on phylogeny and spatial expression of SpAPLs (Figure 6 and 2b), they have their homologs in grass species. SpAPL2 and SpAPL3 are active in turions, while SpAPL1 is expressed at a higher level in fronds. The transcript level of SpAPL2 and SpAPL3 are active at an early phase of turion formation, while all transcript level of SpAPLs decline towards the end phase. It could account for the inhibition of total RNA synthesis after 3 days in ABA, which leads to the shutdown of all primary processes and onset of the dormant state . Analysis of networks of gene expression during Arabidopsis seed filling has also shown that expression of carbohydrates occurred early in seed development . Noticeably, the transcription of SpAPL1 and SpAPL2 is suppressed right after one day of ABA addition, which is quite consistent with previous findings that ABA could inhibit DNA, protein, and RNA synthesis during turion development . But this inhibitory effect of ABA during turion development is selective for that the synthesis of certain turion specific proteins increases . Indeed, the pattern of expression was consistent with a rate-limiting role for this protein in starch biosynthesis. Furthermore, the regulation of gene copies underwent divergence and probably sub-functionalization to permit metabolic differentiation.
In plants, ADP-glucose pyrophosphorylases consist of large and small subunits that share many amino acids due to the proposed origination from a common ancestral gene . For example, APLs and APSs, which make up the heterotetrametic potato enzyme, share significant sequence homology (53% identity and 73% similarity) . Here we selected the large subunit for our analysis because we made the assumption that both are coordinately expressed and that the large subunit should suffice as a marker of the developmental switch between frond and turion stage of the life cycle. Furthermore, the current sequencing of the entire genome will provide an opportunity to locate the gene copies of the small subunit as well. The model structure of the large subunit confirms that N-and C-terminal regions of the SpAPLs are essential for the allosteric regulatory properties of the heterotetrameric enzyme AGPase (Figure 7b) . Even though APLs are considered as a catalytic-disabled subunit, the ability of binding effectors (3-PGA) and substrates (ATP) is likely to undergo a conformational transition similar to the APSs during its catalytic cycle .
Phylogenetic analysis showed that SpAPL1 and SpAPL2 descended from common ancestors of the plastidial form Type 1 and Type 4 of the grasses, respectively, while SpAPL3 shares the same branch with the ancestor of cytosolic Type 2 and plastidial Type 3 of grasses (Figure 6) . Studies suggest that cytosolic Type 2 in grass evolved from a duplication of an ancestral gene encoding a Type 3 plastidial APL by loss-of-function of the transit peptide cleavage site . A similar process might have taken place in Spirodela, where SpAPL1 does not exhibit a clear transit peptide. Interestingly, the opposite seems to be true for SpAPL3, which clusters with cytosolic Type 2 APLs, but encodes a transit peptide. Based on this, we classify it as a plastidial Type 3 APL of the grasses. The phylogenetic relationship will become clearer when we know whether these copies are clustered or dispersed in the Spirodela genome. Interestingly, there is differential invasion of MITES in the introns of these genes with the most pronounced invasion in the SpAPL1 gene (Table 1). This is reminiscent of the grasses, where one of the smallest genomes, rice, had a relative high percentage of MITEs (13.3% of all repeat elements compared to 0.4% in maize), but low retrotransposon content (59.5% compare to 92.7% in maize). Spirodela polyrhiza was namely chosen for sequencing because of its small genome size. Given the genome size variation among Lemnoideae, perhaps a similar relationship of genome size and MITEs exists among Lemnoideae as has been found in grass species .