The Evolutionary Basis of Naturally Diverse Rice Leaves Anatomy

Rice contains genetically and ecologically diverse wild and cultivated species that show a wide variation in plant and leaf architecture. A systematic characterization of leaf anatomy is essential in understanding the dynamics behind such diversity. Therefore, leaf anatomies of 24 Oryza species spanning 11 genetically diverse rice genomes were studied in both lateral and longitudinal directions and possible evolutionary trends were examined. A significant inter-species variation in mesophyll cells, bundle sheath cells, and vein structure was observed, suggesting precise genetic control over these major rice leaf anatomical traits. Cellular dimensions, measured along three growth axes, were further combined proportionately to construct three-dimensional (3D) leaf anatomy models to compare the relative size and orientation of the major cell types present in a fully expanded leaf. A reconstruction of the ancestral leaf state revealed that the following are the major characteristics of recently evolved rice species: fewer veins, larger and laterally elongated mesophyll cells, with an increase in total mesophyll area and in bundle sheath cell number. A huge diversity in leaf anatomy within wild and domesticated rice species has been portrayed in this study, on an evolutionary context, predicting a two-pronged evolutionary pathway leading to the ‘sativa leaf type’ that we see today in domesticated species

High vein frequency is conserved in closely related wild rice but not dependent on the leaf morphological types.

<p>Leaf surface images at the middle of the figure, show increased vein density (white parallel bands) in morphologically diverse leaves (see plant images) of closely related species of HHKK, HHJJ, GG, and FF genomes. Numbers, at the top and below these leaf surface images represent the vein number at 2mm space in case of <i>O</i>. <i>schlechteri</i> (one of the high vein density species) and <i>O</i>. <i>sativa</i> IR64. Scale bar under the leaf surface image = 1 mm. Positions of the veins are marked by red stars (*) in the leaf transverse section (TS) at the right, confirmed that the increased vein frequencies are due to reduced inter-veinal distance (IVD). Leaf types (Sw/Sn), mesophyll (MC) types (A/B), and inter-veinal distance (IVD) are indicated. Sw = Short-wide leaves, Sn = Short-narrow leaves.</p

Leaf anatomical variation in <i>Oryza</i>.

<p>2D leaf anatomical images as viewed in transverse sections. Cell types and the arrangement of cells are as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164532#pone.0164532.g001" target="_blank">Fig 1</a>. Significant variation is noticed for mesophyll cell, bundle sheath cell, and vein size and shape (detailed quantification is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164532#pone.0164532.s005" target="_blank">S2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164532#pone.0164532.s008" target="_blank">S5</a> Tables) in the <i>Oryza</i> family. <i>Oryza coarctata</i> and <i>O</i>. <i>australiensis</i> have the thickest leaves among the species. In contrast, species of the GG, HHJJ, HHKK, and BB genomes have thinner leaves. In addition, the leaves of the species of the HHKK, HHJJ, and GG genomes show closer vein spacing with relatively smaller mesophyll cells. Notably, the <i>O</i>. <i>coarctata</i> leaf possesses the widest bundle sheath cell and vertically placed additional veins unique among the rest of the <i>Oryza</i> species. Scale bar = 50 μm.</p

Rice leaf evolution.

<p>A two-pronged leaf evolutionary hypothesis in rice suggests that the leaf structure has possibly evolved into <i>Oryza sativa</i> leaf type by the favorable selection of one of the two possible evolutionary lines (Lineage 1 and 2). The first lineage explains the presence of Type-A mesophyll cells, which existed ~10 million years ago and has remained unchanged since the evolution of <i>O</i>. <i>schlechteri</i>. The second lineage explains a gradual modification of the ancestral Type-B mesophyll cells that lead to an overall increase in the mesophyll area between the veins and gradually evolved into the cultivated rice leaf that we see today. The probable time of evolution of a particular leaf type is shown as Million Years Ago (MYA).</p

3-Dimensional anatomical models of plolyploid <i>Oryza</i> rice leaves.

<p>The 3D models were constructed by combining the measurements taken separately along the three growth axes (X, Y, and Z) for all polyploid <i>Oryza</i> species. Note that, when considering the perpendicular spatial positioning of the mesophyll cell and bundle sheath cell to each other; X, Y, and Z represent length, height, and width of mesophyll cell, and the width, height, and length of bundle sheath cell respectively. Mesophyll cells are colored in green to represent the main photosynthetic tissue, veins are colored in light green, and bundle sheath cells are colored gray. Wavy surfaces are applied to the mesophyll cell boundaries with a degree of lobing value more than 1.1 (Type-B mesophyll cell). Type-A mesophyll cell is shown with smooth wall structure as in <i>O</i>. <i>schlechteri</i>, <i>O</i>. <i>longiglumis</i>, <i>O</i>. <i>ridleyi</i>. Genome types are as described in the text. A calibration scale of 50 μm is provided.</p

A schematic representation of a rice leaf shows different cell types and three different leaf dissection axes; used to calculate cell size, number, and volume.

<p>For ease of viewing, bulliform and epidermal cells are not shown in detail. (A) Rice leaf transverse section. The position of major leaf cells: mesophyll cell (MC, coloured green), bundle sheath cells (BSC, coloured white), vein (V, coloured blue), bulliform cells (BL), stone cells (ST), and epidermal layers. In rice, inter-veinal distance is filled by a number of elliptic mesophyll cells. Veins are surrounded by a wreath of bundle sheath cells and crowned by bundle sheath cell extension above the main circle. Large bulliform cells are present only at the adaxial side of the leaf. Stone cells are present at both the abaxial and adaxial end of the vein. X, Y and Z represent the three growth axes where, X represents the leaf lateral axis, Y represents the leaf abaxial-adaxial axis, and Z represents the leaf longitudinal or the proximo-distal axis. The long axis of the mesophyll cell is perpendicular to the vein axis. The long axis of bundle sheath cell is parallel to the vein axis and perpendicular to the mesophyll cell. LT = Leaf thickness; IVD = Inter-veinal distance; TML = Total mesophyll length in inter-veinal space; VW = Vein width; VH = Vein height. (B) Mesophyll and bundle sheath cell parameters, measured along X, Y, and Z. MCL = mesophyll cell length; MCH = mesophyll cell height; MCW = Mesophyll cell width; and BSCW = Bundle sheath cell width; BSCH = Bundle sheath cell height; and BSCL = Bundle sheath cell length.</p

Variation in mesophyll cell size and lobing.

<p>(A) Leaf transverse sections and longitudinal section to show the mesophyll cell length, mesophyll cell lobing and mesophyll cell width in IR64 and three wild rice species: <i>O</i>. <i>schlechteri</i>, <i>O</i>. <i>granulata</i>, and <i>O</i>. <i>meyeriana</i>. The lobed/smooth line of the mesophyll cell wall (arrows) is false colored in green that is visible as a result of auto-fluorescence of the wall components. Scale bars show 10 μm distance for transverse sections and 20 μm distance for the longitudinal sections. (B) The graph shows the quantitative values (average ±SD) of MCL, MCW, and LB_MC (secondary axis). MCL = mesophyll cell length, MCW = mesophyll cell width, LB_MC = mesophyll cell lobing.</p

Similar leaf morphology or anatomy appears in diverse rice genomes.

<p>(A) <i>Oryza alta</i> (CCDD) and <i>O</i>. <i>longistaminata</i> (AA) show similar Long-wide leaf morphologies but quite different in their leaf anatomies. (B) Similar mesophyll cell numbers (4–5, marked by the red stars in leaf transverse sections) appear in <i>O</i>. <i>coarctata</i>, <i>O</i>. <i>grandiglumis</i>, and <i>O</i>. <i>minuta</i> (KKLL, CC, and BBCC genome respectively) in spite of their markedly different leaf types. Lw = Long-wide, Sn = Short-narrow, Sw = Short-wide.</p

Ancestral state reconstruction of leaf morphology and anatomical traits in diploid <i>Oryza</i> species.

<p>The history of the evolution of leaf traits in diploid species confirms the increase in the inter-veinal mesophyll area, mesophyll number, mesophyll length and bundle sheath cell number over time in rice. Leaf thickness and bundle sheath cell width also appear to be reduced in the recently evolved rice species.</p

Ancestral state reconstruction of leaf morphology and anatomy traits.

<p>Historical analysis of a total of 13 leaf traits, taking all the <i>Oryza</i> species, confirm that small leaf, high vein density, shorter inter-veinal mesophyll area, smaller-sized mesophyll cells, and fewer number of bundle sheath cells surrounding a vein, are the primitive leaf characters in rice. Likewise, a wider inter-veinal mesophyll area, highly-lobed mesophyll cells, and increased bundle sheath cell numbers are advanced characters in cultivated rice leaves. <i>Rhynchoryza subulata</i> was used as an out-group.</p