Fig 1.
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.
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.
Fig 2.
Leaf shape diversity in Oryza.
Cluster analysis of species based on leaf blade length and leaf blade width generates two distinct groups of long and short leaved species, represented with a green background for long leaves and a yellow background for short leaves. Each species name is further accompanied with its genome type as mentioned in the text. The bar graphs show leaf blade width of long and short leaves separately to show the narrow and wide types. Representative photos of four leaf types in Oryza are shown as A = Long-wide (Lw), B = Long-narrow (Ln), C = Short-wide (Sw), and D = Short-narrow (Sn) leaf. Respective leaf types of Oryza species are mentioned in the black and white shaded boxes together with their preferred growing habitat, i.e., sunny/shaded. Notably, majority of the species in the first cluster are sun loving, whereas most of the species in the second cluster are shade loving. This suggests that sun-loving species mostly have long leaves whereas shade-loving species generally have short leaves.
Fig 3.
Leaf anatomical variation in Oryza.
2D leaf anatomical images as viewed in transverse sections. Cell types and the arrangement of cells are as described in Fig 1. Significant variation is noticed for mesophyll cell, bundle sheath cell, and vein size and shape (detailed quantification is given in S2–S5 Tables) in the Oryza family. Oryza coarctata and O. australiensis 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 O. coarctata leaf possesses the widest bundle sheath cell and vertically placed additional veins unique among the rest of the Oryza species. Scale bar = 50 μm.
Fig 4.
Variation in mesophyll cell size and lobing.
(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: O. schlechteri, O. granulata, and O. meyeriana. 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 LBMC (secondary axis). MCL = mesophyll cell length, MCW = mesophyll cell width, LBMC = mesophyll cell lobing.
Fig 5.
High vein frequency is conserved in closely related wild rice but not dependent on the leaf morphological types.
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 O. schlechteri (one of the high vein density species) and O. sativa 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.
Fig 6.
Similar leaf morphology or anatomy appears in diverse rice genomes.
(A) Oryza alta (CCDD) and O. longistaminata (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 O. coarctata, O. grandiglumis, and O. minuta (KKLL, CC, and BBCC genome respectively) in spite of their markedly different leaf types. Lw = Long-wide, Sn = Short-narrow, Sw = Short-wide.
Fig 7.
3-Dimensional anatomical models of plolyploid Oryza rice leaves.
The 3D models were constructed by combining the measurements taken separately along the three growth axes (X, Y, and Z) for all polyploid Oryza 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 O. schlechteri, O. longiglumis, O. ridleyi. Genome types are as described in the text. A calibration scale of 50 μm is provided.
Fig 8.
3-Dimensional anatomical models of diploid Oryza rice leaves.
3D models describe the variation of rice leaf cellular structure of all diploid Oryza species; especially describing the characters of mesophyll cells, bundle sheath cells and veins. The construction of the 3D models is as described as in Fig 7. Type-A mesophyll cell is shown with smooth wall structure as in O. meyeriana and O. granulata. Three Oryza sativa cultivars IR64, IR24 and IR31917 are shown to provide a broad idea about the leaf anatomy of present day cultivated rice species. Genome types are as described in the text. A calibration scale of 50 μm is provided.
Fig 9.
Ancestral state reconstruction of leaf morphology and anatomy traits.
Historical analysis of a total of 13 leaf traits, taking all the Oryza 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. Rhynchoryza subulata was used as an out-group.
Fig 10.
Ancestral state reconstruction of leaf morphology and anatomical traits in diploid Oryza species.
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.
Fig 11.
A two-pronged leaf evolutionary hypothesis in rice suggests that the leaf structure has possibly evolved into Oryza sativa 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 O. schlechteri. 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).