Distribution patterns of idioblast clusters on the adaxial and abaxial surfaces of leaves.

<p>(A) Fluorescence images of the adaxial and abaxial surfaces of half-leaves under UV excitation. Scale bar: 1 mm. (B) Schematic of the spatial distribution of singlet (gray), doublet (green), triplet (red), and quadruplet and more (orange) idioblasts across the half-leaves. (C) Frequency of idioblast clusters on the whole adaxial and abaxial surfaces of leaves. Data collected from 18 leaves for the adaxial (top) or 19 leaves for the abaxial (bottom) surface are shown. (D, E) Frequency of idioblast clusters on the whole adaxial and abaxial surfaces of leaves. Data from individual leaves for the adaxial (D) and the abaxial (E) surfaces are shown.</p

Relationship between the leaf surface area and the number of idioblast clusters or cells.

<p>(A) Idioblast clusters. (B) Idioblast cells. Data from 18 leaves for the adaxial (open symbols) or 19 leaves for the abaxial (filled symbols) surfaces are plotted.</p

Presence and mobility of boundaries defining areas of idioblast formation on both leaf surfaces.

<p>(A) Schematics of adaxial (cyan dots in the colored areas) and abaxial (color annotations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118965#pone.0118965.g004" target="_blank">Fig. 4</a>) idioblast clusters in three individual, whole leaves. (B–D) Fluorescence images of the apical (B), central (C), and basal (D) leaf zones under UV excitation. Scale bars: 2 mm (A); 200 μm (B–D).</p

Plastid phenotypes in leaf PCs of <i>Arabidopsis</i> WT, <i>arc5</i>, <i>arc6</i>, and <i>atminE1</i>.

<p>(A–J) Images of plastid-targeted YFP in the third-fourth leaf petioles of 4-week-old WT (A), <i>arc5</i> (B, E, H), <i>arc6</i> (C, F, I), and <i>atminE1</i> (D, G, J) seedlings. CLSM images of maximal intensity projection are shown. Giant plastids (arrows), putative dividing plastids (arrowheads), stomata (double arrowheads), and mini-plastids (asterisks) are indicated. Bar = 10 μm.</p

Plastid phenotypes in leaf stomatal GCs in <i>Arabidopsis</i> WT, <i>arc5</i>, <i>arc6</i>, and <i>atminE1</i> plants.

<p>(A–I) Images of plastid-targeted YFP in the third-fourth leaf petioles of 4-week-old seedlings from the WT (A), <i>arc5</i> (B–D), <i>arc6</i> (E–G), and <i>atminE1</i> (H, I). CLSM images of maximal intensity projection (top panels) and merged with DIC (bottom panels) are shown. (J, K) Images of YFP-labeled plastids in GCs reconstructed from a series of optical sections generated by CLSM, taken at 0.3 or 0.6 μm intervals. The GC pairs in panels (J) and (K) are identical to those in panels (D) and (G), respectively. (L) Epifluorescence microscopy images of GCs in the third-fourth leaf petioles of 4-week-old seedlings from <i>arc6</i>. Images of YFP (top panels) and DIC (bottom panels) are shown. Bar = 10 μm.</p

UV-induced autofluorescence from idioblasts in mature and late expanding leaves.

<p>(A–D) Bright-field or fluorescent images of idioblasts in mature (A, B) and late-developing (C, D) leaves. For fluorescence microscopy, cells were excited with UV (330–385 nm) and observed at >420 nm (A–D), 417–477 nm, 467–499 nm, >510 nm, 518–572 nm or >575 nm (B). Scale bars: 50 μm.</p

Organ-Level Analysis of Idioblast Patterning in <i>Egeria densa</i> Planch. Leaves

<div><p>Leaf tissues of plants usually contain several types of idioblasts, defined as specialized cells whose shape and contents differ from the surrounding homogeneous cells. The spatial patterning of idioblasts, particularly of trichomes and guard cells, across the leaf epidermis has received considerable attention as it offers a useful biological model for studying the intercellular regulation of cell fate and patterning. Excretory idioblasts in the leaves of the aquatic monocotyledonous plant <i>Egeria densa</i> produced light blue autofluorescence when irradiated with ultraviolet light. The use of epifluorescence microscopy to detect this autofluorescence provided a simple and convenient method for detecting excretory idioblasts and allowed tracking of those cells across the leaf surfaces, enabling quantitative measurement of the clustering and spacing patterns of idioblasts at the whole leaf level. Occurrence of idioblasts was coordinated along the proximal–distal, medial–lateral, and adaxial–abaxial axes, producing a recognizable consensus spatial pattern of idioblast formation among fully expanded leaves. Idioblast clusters, which comprised up to nine cells aligned along the proximal–distal axis, showed no positional bias or regularity in idioblast-forming areas when compared with singlet idioblasts. Up to 75% of idioblasts existed as clusters on every leaf side examined. The idioblast-forming areas varied between leaves, implying phenotypic plasticity. Furthermore, in young expanding leaves, autofluorescence was occasionally detected in a single giant vesicle or else in one or more small vesicles, which eventually grew to occupy a large portion of the idioblast volume as a central vacuole. Differentiation of vacuoles by accumulating the fluorescence substance might be an integral part of idioblast differentiation. Red autofluorescence from chloroplasts was not detected in idioblasts of young expanding leaves, suggesting idioblast differentiation involves an arrest in chloroplast development at a very early stage, rather than transdifferentiation of chloroplast-containing epidermal cells.</p></div

Plant and leaf structures of <i>Egeria densa</i>.

<p>(A) Plant stature. (B) Three cell types in the mature leaf epidermis. Epidermal cells (arrowheads), idioblasts (arrows) and a marginal prickle-hair or tooth cell (double arrowhead) are represented. Note that the top surfaces of the projected idioblasts are in focus in this image. (C) Cross-section of the central zone of the mature leaf. The adaxial and abaxial sides of the leaf and the midrib are indicated. Inset represents magnification of the boxed area and shows an idioblast (arrow). (D) Bright-field (BF) and ultraviolet (UV)-induced fluorescence images of the leaf epidermis (taken using 10× objective). (E) Fluorescence image of the UV-irradiated leaf epidermis (taken using 4× objective). Scale bars: 5 mm (A); 50 μm (B–D); 200 μm (E).</p

Grape-like plastid clusters in leaf PCs of <i>arc6</i>.

<p>Images of plastid-targeted YFP in the third-fourth leaf petioles of 4-week-old seedlings from <i>arc6</i> are shown. (A) Formation of plastid bulges. (B) Serial optical sections of grape-like plastid clusters. Arrowheads indicate the activated regions of plastid bulges. Bar = 10 μm.</p

The <i>Arabidopsis arc5</i> and <i>arc6</i> mutations differentially affect plastid morphology in pavement and guard cells in the leaf epidermis

<div><p>Chloroplasts, or photosynthetic plastids, multiply by binary fission, forming a homogeneous population in plant cells. In <i>Arabidopsis thaliana</i>, the division apparatus (or division ring) of mesophyll chloroplasts includes an inner envelope transmembrane protein ARC6, a cytoplasmic dynamin-related protein ARC5 (DRP5B), and members of the FtsZ1 and FtsZ2 families of proteins, which co-assemble in the stromal mid-plastid division ring (FtsZ ring). FtsZ ring placement is controlled by several proteins, including a stromal factor MinE (AtMinE1). During leaf mesophyll development, <i>ARC6</i> and <i>AtMinE1</i> are necessary for FtsZ ring formation and thus plastid division initiation, while <i>ARC5</i> is essential for a later stage of plastid division. Here, we examined plastid morphology in leaf epidermal pavement cells (PCs) and stomatal guard cells (GCs) in the <i>arc5</i> and <i>arc6</i> mutants using stroma-targeted fluorescent proteins. The <i>arc5</i> PC plastids were generally a bit larger than those of the wild type, but most had normal shapes and were division-competent, unlike mutant mesophyll chloroplasts. The <i>arc6</i> PC plastids were heterogeneous in size and shape, including the formation of giant and mini-plastids, plastids with highly developed stromules, and grape-like plastid clusters, which varied on a cell-by-cell basis. Moreover, unique plastid phenotypes for stomatal GCs were observed in both mutants. The <i>arc5</i> GCs rarely lacked chlorophyll-bearing plastids (chloroplasts), while they accumulated minute chlorophyll-less plastids, whereas most GCs developed wild type-like chloroplasts. The <i>arc6</i> GCs produced large chloroplasts and/or chlorophyll-less plastids, as previously observed, but unexpectedly, their chloroplasts/plastids exhibited marked morphological variations. We quantitatively analyzed plastid morphology and partitioning in paired GCs from wild-type, <i>arc5</i>, <i>arc6</i>, and <i>atminE1</i> plants. Collectively, our results support the notion that ARC5 is dispensable in the process of equal division of epidermal plastids, and indicate that dysfunctions in ARC5 and ARC6 differentially affect plastid replication among mesophyll cells, PCs, and GCs within a single leaf.</p></div