Characterization of active miniature inverted-repeat transposable elements in the peanut genome

Miniature inverted-repeat transposable elements (MITEs), some of which are known as active non-autonomous DNA transposons, are found in the genomes of plants and animals. In peanut (Arachis hypogaea), AhMITE1 has been identified in a gene for fatty-acid desaturase, and possessed excision activity. However, the AhMITE1 distribution and frequency of excision have not been determined for the peanut genome. In order to characterize AhMITE1s, their genomic diversity and transposition ability was investigated. Southern blot analysis indicated high AhMITE1 copy number in the genomes of A. hypogaea, A. magna and A. monticola, but not in A. duranensis. A total of 504 AhMITE1s were identified from the MITE-enriched genomic libraries of A. hypogaea. The representative AhMITE1s exhibited a mean length of 205.5 bp and a GC content of 30.1%, with AT-rich, 9 bp target site duplications and 25 bp terminal inverted repeats. PCR analyses were performed using primer pairs designed against both flanking sequences of each AhMITE1. These analyses detected polymorphisms at 169 out of 411 insertional loci in the four peanut lines. In subsequent analyses of 60 gamma-irradiated mutant lines, four AhMITE1 excisions showed footprint mutations at the 109 loci tested. This study characterizes AhMITE1s in peanut and discusses their use as DNA markers and mutagens for the genetics, genomics and breeding of peanut and its relatives

Transformation of lipid bodies related to hydrocarbon accumulation in a green alga, Botryococcus braunii (Race B).

The colonial microalga Botryococcus braunii accumulates large quantities of hydrocarbons mainly in the extracellular space; most other oleaginous microalgae store lipids in the cytoplasm. Botryococcus braunii is classified into three principal races (A, B, and L) based on the types of hydrocarbons. Race B has attracted the most attention as an alternative to petroleum by its higher hydrocarbon contents than the other races and its hydrocarbon components, botryococcenes and methylsqualenes, both can be readily converted into biofuels. We studied race B using fluorescence and electron microscopy, and clarify the stage when extracellular hydrocarbon accumulation occurs during the cell cycle, in a correlation with the behavior and structural changes of the lipid bodies and discussed development of the algal colony. New accumulation of lipids on the cell surface occurred after cell division in the basolateral region of daughter cells. While lipid bodies were observed throughout the cell cycle, their size and inclusions were dynamically changing. When cells began dividing, the lipid bodies increased in size and inclusions until the extracellular accumulation of lipids started. Most of the lipids disappeared from the cytoplasm concomitant with the extracellular accumulation, and then reformed. We therefore hypothesize that lipid bodies produced during the growth of B. braunii are related to lipid secretion. New lipids secreted at the cell surface formed layers of oil droplets, to a maximum depth of six layers, and fused to form flattened, continuous sheets. The sheets that combined a pair of daughter cells remained during successive cellular divisions and the colony increased in size with increasing number of cells

EM analysis of lipid bodies after septum formation to just completing cell wall formation.

<p><b>A</b>. Longitudinal section of dividing cells just after septum formation but before cell wall formation. <b>B</b>. Cross section of septum developing cell. <b>C</b>. Lipid bodies contact to electron-dense bodies enclosed unit membrane. <b>D</b>. A pair of daughter cells during cell wall formation. Note that all lipid bodies in the earlier stage (A) are filled with a similar electron-dense material but in D they became patchy with emergence of electron-dense bodies. ch, chloroplast; G, Golgi body; m-CW, mother cell wall; N, nucleus; n-CW, new cell wall; arrowhead, electron-dense body enclosed by unit membrane; white arrow, septum; * lipid body; arrowhead, electron-dense body enclosed by unit membrane. Scale bars in A,B,D and C: 1 µm and 0.5 µm, respectively.</p

Colony double-stained with Nile red and neutral red.

<p><b>A</b>. Neutral red, bright-field microscopy. <b>B</b>. Nile red, fluorescence microscopy. Numbers 1–5 with arrows are indicating representative cells or cell pairs at the specific stages of cell cycle as 1; interphase cell, 2; growing cell ∼ cells just after septum formation, 3; cells accumulating lipids between the septum and on the cell surface at basolateral region, 4; a pair of immature daughter cells whose cell area is <2/3 of interphase cell, and 5; A pair of mature daughter cells whose cell area is ≥2/3 of interphase cell. *, exuded lipids from the extracellular matrix. <b>C</b>. Three typical staining patterns seen for cells at the stage 3. <b>3-1∼3A</b>. Neutral red, <b>3-1∼3B</b>. Nile red. <b>3-1</b>. A dividing cell with lipid bodies accumulates lipids at the edge of the septum. <b>3-2</b>. A dividing cell with a reduced number of lipid bodies accumulates lipids around the septum. <b>3-3</b>. A dividing cell without lipid bodies filled lipids around the septum. Lipids on the cell surface at basolateral region are also accumulated for these cells. Arrowhead, newly accumulating lipids on the cell surface at basolateral region; white arrow, septum. <b>D</b>. Changes in the total area of lipid bodies (red diamond) and vacuoles (blue circle) per cell. Stage number is corresponding to the number in <b>A</b>/<b>B</b>. For each stage, the average area of the lipid bodies (LB) and vacuoles (V) per cell are shown (<i>n</i> = 50 for each stage, error bars indicate standard deviation). The total area of lipid bodies per cell differed significantly (<i>p</i><0.01) among the five stages, except for between stage 4 and 5. Scale bars in A, B and C: 10 µm and 2 µm, respectively.</p

Transformation of lipid bodies during the cell cycle.

<p>Red solid line, increase in volume of lipid bodies; red dot line, keeping in volume of lipid bodies; blue solid line, decrease in volume of lipid bodies.</p

EM analysis of lipid bodies in cells from interphase to early septum formation.

<p><b>A</b>. Longitudinal section of interphase cell. The nucleus located at the center and a cup-shaped chloroplast occupies the bottom and side region, large lipid bodies are seen between them. The Golgi apparatus is at cell apex. <b>B</b>. Tangential section of early growing cell with swelling extracellular space at the apical region. <b>C</b>. Cross section of metaphase cell. <b>D</b>. Comparison of limiting membrane between lipid body (lipid monolayer) and vacuole (lipid bilayer). <b>E</b>. Tangential section at apical region of cell at early septum formation. <b>E2</b>. Enlargement of E1. C, chromosome; ch, chloroplast; CW, cell wall; ER, endoplasmic reticulum; G, Golgi body; N, nucleus; m-CW, mother cell wall; P, pyrenoid; PM, plasma membrane; V, vacuole; * lipid body; thick arrow, contact site between lipid body and the ER; thin black arrow, thin layer; white arrow, septum. Scale bars in A-C,E1 and D,E2: 1 µm, 0.5 µm and 0.1 µm, respectively.</p

Colony of <i>B. braunii</i> race B by electron microscopy.

<p><b>A</b>. Sample frozen in liquid propane, <b>B</b>. Sample frozen by high-pressure freezing machine. <b>A1</b>. Each colony is enclosed by colony sheath (double ended arrow). <b>A2</b>. Enlargement of the rectangle in A1. Colony sheath is composed of fibrils stretching from the apical region of each cell and the upper edge of the electron-dense intercellular matrix. Inset is the enlargement of a part of A2. <b>B1</b>. Each cell is covered with 3–6 electron-dense thin layers in the basolateral region. These layers are appeared to be holding the colonial cells together. <b>B2</b>. Enlargement of the rectangle in B1 (small arrow indicates each electron-dense thin layer). <b>C</b>. A speculative genealogic relationship among the cells in the colony shown in B1. ch, chloroplast; N, nucleus; *, lipid body. Scale bars in A1 and A2–B2: 10 µm and 1 µm, respectively.</p