Anatomy and ultrastructure of embryonic leaves of the C4 species Setaria viridis

Anatomy and ultrastructure of embryonic leaves of the C4 species Setaria viridis Abstract Background and Aims Setaria viridis is being promoted as a model C4 photosynthetic plant because it has a small genome (~515 Mb), a short life cycle (~60 d) and it can be transformed. Unlike other C4 grasses such as maize, however, there is very little information about how C4 leaf anatomy (Kranz anatomy) develops in S. viridis. As a foundation for future developmental genetic studies, we provide an anatomical and ultrastructural framework of early shoot development in S. viridis, focusing on the initiation of Kranz anatomy in seed leaves. Methods Setaria viridis seeds were germinated and divided into five stages covering development from the dry seed (stage S0) to 36 h after germination (stage S4). Material at each of these stages was examined using conventional light, scanning and transmission electron microscopy. Key Results Dry seeds contained three embryonic leaf primordia at different developmental stages (plastochron 1–3 primordia). The oldest (P3) leaf primordium possessed several procambial centres whereas P2 displayed only ground meristem. At the tip of P3 primordia at stage S4, C4 leaf anatomy typical of the malate dehydrogenase-dependent nicotinamide dinucleotide phosphate (NADP-ME) subtype was evident in that vascular bundles lacked a mestome layer and were surrounded by a single layer of bundle sheath cells that contained large, centrifugally located chloroplasts. Two to three mesophyll cells separated adjacent vascular bundles and one mesophyll cell layer on each of the abaxial and adaxial sides delimited vascular bundles from the epidermis. Conclusions The morphological trajectory reported here provides a foundation for studies of gene regulation during early leaf development in S. viridis and a framework for comparative analyses with other C4 grasses. C4, Setaria viridis, Kranz anatomy, embryonic leaves, vascular development INTRODUCTION C4 photosynthesis occurs in 19 angiosperm families that encompass both monocots and eudicots, with monocot representatives including ~4500 grass species (Sage et al., 2011; Williams et al., 2012). The C4 pathway differs from the C3 pathway in that photosynthesis is split between two cell types. CO2 is initially fixed in the mesophyll (M) cells by phosphoenolpyruvate carboxylase and then after decarboxylation of a C4 acid, it is re-fixed by ribulose bisphosphate carboxylase/oxygenase in the bundle sheath (BS) cells (Von Caemmerer and Furbank, 2003; Langdale, 2011). In most C4 plants, BS and M cell types are arranged around leaf veins in a characteristic anatomy known as Kranz (Brown, 1975; Dengler et al., 1985). Although Kranz anatomy is not a prerequisite for C4 photosynthesis (Voznesenskaya et al., 2001; Sage, 2002), it is present in all C4 grasses. C4 plants have previously been classified into three subtypes, depending on the mechanism by which C4 acids are decarboxylated in the BS cells. Although the biochemical distinction between these subtypes is now questioned (Furbank, 2011), there are distinct differences between BS cell anatomy in so-called malate dehydrogenase-dependent nicotinamide dinucleotide phosphate (NADP-ME), NAD-ME and PEP-CK subtypes (Brown, 1975; Edwards and Voznesenskaya, 2011). In NADP-ME species, BS cell chloroplasts lack well-developed grana and are located centrifugally; in NAD-ME species, BS chloroplasts are positioned centripetally and possess well-developed grana; and in PEP-CK species, BS chloroplasts possess well-developed grana and are arranged in a centrifugal position. In addition to differences in chloroplast structure and arrangement, BS cells are distinguished from M cells by a range of other features including number, arrangement and size of mitochondria, degree of vacuolation and type of plasmodesmata. The thickness and composition of the cell wall can also differ (Dengler et al., 1986; Edwards and Voznesenskaya, 2011). For example, the BS cells of several species in the NADP-ME and PEP-CK subgroups have a suberized lamella (Eastman, 1988; Edwards and Voznesenskaya, 2011). With at least 62 lineages of C4 plants now identified (Sage et al., 2011), further variation in structure and organization of Kranz anatomy is likely to be discovered (Williams et al., 2012), reinforcing the need for a clear and detailed understanding of both anatomy and metabolism in species used as C4 models. The C4 grass Setaria viridis has been proposed as a model for understanding C4 metabolism and anatomy (Brutnell et al., 2010; Diao et al., 2014), with the genome sequence of closely related S. italica (Bennetzen et al., 2012) and transformation protocols (Martins et al., 2015; Van Eck and Swartwood, 2015; Saha and Blumwald, 2016) opening the way for comprehensive analyses of C4 photosynthesis in a rapid cycling plant. Setaria viridis is an NADP-ME C4 grass (Aliscioni et al., 2016), but is phylogenetically related to switchgrass (NAD-ME subtype) and Panicum virgatum (PEP-CK subtype) (Brutnell et al., 2010). Six subclasses of NADP-ME subtype anatomy have been identified in Poaceae, including the most common ‘Classic’ subtype (Edwards and Voznesenskaya, 2011). The different anatomies mainly vary with respect to (1) presence or absence of the mestome sheath and suberized lamella, (2) number of BS cell layers and (3) ultrastructure of chloroplasts (Edwards and Voznesenskaya, 2011). Because photosynthetic BS cells are a defining feature of C4 plants, understanding the ontogeny of this cell type is central to any interpretation of the function of these different anatomical arrangements and of the relationship between species possessing them. We have therefore conducted a detailed analysis of the structure and the ultrastructure of BS cells in embryonic leaves of S. viridis during germination, from seed imbibition to coleoptile rupture by the first leaf. The data presented here on the pattern of Kranz differentiation in S. viridis seed leaves provide a platform for future morphological and comparative studies with this new C4 model species. MATERIALS AND METHODS Plant growth and sampling Fruit of Setaria viridis (L.) Beauvois accession A10.1 were collected from greenhouse-grown plants at the Universidade Federal do Rio de Janeiro - UFRJ (Brazil) in January 2015 and stored at room temperature (25–28 °C) for 10 months before use. Prior to sampling for microscopy, ~150 fruits were placed in 30 mm potassium nitrate solution for 24 h at room temperature and then washed three times with gentle agitation in distilled water (modified from Sebastian et al., 2014). Germination was then carried out on moistened Germitest paper (Germilab, Brazil) in Petri dishes in a BOD incubator (Eletrolab EL202, SP, Brazil) at 28 °C under continuous light. For light and transmission electron microscopy (TEM), ten fruits with bracts were sampled at each of the sampling times: 0 h (dry seed), 12 h, 15 h, 24 h and 36 h after imbibition. Sampling times were established based on pilot studies that identified morphological landmarks such as the protrusion of coleorhiza and the appearance of the primary root. The developmental stage of the seed at each sampling time was classified as S0 (0 h), S1 (12 h), S2 (15 h), S3 (24 h) and S4 (36 h). Seeds became swollen between stages S0 and S1, and the coleorhiza emerged at stage Sl. Stage S2 was characterized by the presence of hairs on the coleorhiza and stage S3 by the appearance of the coleoptile and the primary root. By stage S4, the first embryonic leaf had ruptured the coleoptile. Five fruits without bracts were collected for scanning electron microscopy (SEM) at the same sampling times. All bracts (two glumes, sterile lemma, fertile lemma and palea) were removed from the fruits to facilitate SEM study of post-germination events. To examine the overall venation pattern in expanded leaves, the first leaf to emerge was harvested around 1 week after seed imbibition. The leaf was cleared with ethanol at 90 °C and stained in 1 % safranin (modified from Kraus and Arduin, 1997). Anatomical and ultrastructural studies Plant material was fixed in 4 % paraformaldehyde and 2.5 % glutaraldehyde in 0.1 m phosphate buffer for 24 h and post-fixed in 1 % osmium tetroxide in 0.1 m phosphate buffer for 1 h. For SEM, fruits without bracts were then dehydrated through an ethanol gradient, critical-point dried and sputter-coated with gold (Vega 3LMU Tescan, Brno, Czech Republic). Samples were viewed using a scanning electron microscope (Spirit Biotwin 12, FEI Company, Hillsboro, OR, USA). For light microscopy, fruits with bracts were dehydrated in an ethanol/propylene oxide series, embedded in Spurr’s resin (Premix Kit-Hard, TAAB Laboratory and Microscopy, Aldermaston, UK) and polymerized at 70 °C overnight. Transverse thin sections (1 µm) of the embedded material were cut from the tip to the base of the coleoptile using an ultramicrotome (RMC Products, Tucson, AZ, USA), stained with 0.05 % toluidine blue and viewed in a Leica DM 2500 (Wetzlar, Germany) optical microscope. Sequential 1-µm sections were used to create longitudinal diagrams of the leaf vascular pattern based on the structures present at each developmental stage. For TEM, ultrathin sections (60 nm) were cut using an ultramicrotome (ultra-RMC Products), mounted on copper grids, and contrasted with 1 % uranyl acetate in water for 25 min followed by lead citrate for 3 min. Sections were examined in a transmission electron microscope (Spirit Biotwin 12 FEI Company). Measurements were carried out using the TEM Imaging Platform program. RESULTS General development during the first 36 h of germination SEM images of the diaspore (dispersion unit) of S. viridis revealed a caryopsis consisting of five bracts – two glumes, a sterile and a fertile lemma, plus a palea (Stage S0; Fig. 1A). The coleorhiza ruptured the seed coat after 12 h of imbibition (Stage Sl; Fig. 1B) and after 15 h the absorbent hairs of the coleorhiza were visible (Stage S2; Fig. 1C). Disruption of the caryopsis as a result of emergence of the coleoptile and the radicle occurred 24 h after imbibition (Stage S3; Fig. 1D), and 36 h after imbibition the first juvenile leaf ruptured the coleoptile (Stage S4; Fig. 1E, F). The diaspore is hereafter referred to as the seed. Fig. 1. View largeDownload slide Scanning electron microscopy of S. viridis diaspore during germination from dry seed to 36 h after imbibition (first embryonic leaf rupturing the coleoptile). (A) Stage S0 – dry seed – with bracts: first glume (*), second glume (**), sterile lemma (arrow) and fertile lemma (dashed arrow) enveloping the caryopsis (the palea remains covered by the sterile lemma). (B) Stage S1 – 12 h after imbibition, the coleorhiza rupturing the caryopsis (arrow). (C) Stage S2 – 15 h after imbibition, absorbent hairs of the coleorhiza (arrow) are visible. (D) Stage S3 – 24 h after imbibition, coleoptile breaking through the caryopsis (arrow); the radicle (*), the mesocotyl (dashed arrow) and coleorhiza (**) are visible. (E) Stage S4 – 36 h after imbibition, left: intact coleoptile; right: the first juvenile leaf has ruptured the coleoptile (arrow). (F) Ruptured coleoptile shown in detail. Scale bar = 500 μm (A, B, D), 1mm (C), 2 mm (E), 200 μm (F). Fig. 1. View largeDownload slide Scanning electron microscopy of S. viridis diaspore during germination from dry seed to 36 h after imbibition (first embryonic leaf rupturing the coleoptile). (A) Stage S0 – dry seed – with bracts: first glume (*), second glume (**), sterile lemma (arrow) and fertile lemma (dashed arrow) enveloping the caryopsis (the palea remains covered by the sterile lemma). (B) Stage S1 – 12 h after imbibition, the coleorhiza rupturing the caryopsis (arrow). (C) Stage S2 – 15 h after imbibition, absorbent hairs of the coleorhiza (arrow) are visible. (D) Stage S3 – 24 h after imbibition, coleoptile breaking through the caryopsis (arrow); the radicle (*), the mesocotyl (dashed arrow) and coleorhiza (**) are visible. (E) Stage S4 – 36 h after imbibition, left: intact coleoptile; right: the first juvenile leaf has ruptured the coleoptile (arrow). (F) Ruptured coleoptile shown in detail. Scale bar = 500 μm (A, B, D), 1mm (C), 2 mm (E), 200 μm (F). Anatomy of the mature embryo The embryo of mature seeds averages ~800 µm in length and ~150 µm in width, excluding the coleoptile. The shoot apical meristem (SAM) and young leaf primordia are subtended by the mesocotyl, which is characterized by cells that are more densely cytoplasmic than in the SAM, and by the radicle in which longitudinal cell files are apparent. Three leaf primordia are present around the SAM (Fig. 2), referred to as plastochrons (P) 1–3, where a plastochron is the time interval between initiation of sequential primordia. The youngest (P1) primordium has just initiated whereas the P2 and P3 primordia enclose the SAM, as is typical of grass leaf primordia. During the period 0–36 h examined here, no new leaf primordia were initiated at the SAM. Fig. 2. View largeDownload slide Three leaf primordia are evident at the shoot apical meristem (asterisk). The oldest (P3) and P2 primordia encircle the meristem, whereas the youngest (P1) has just been initiated. Scale bar = 20 μm. Fig. 2. View largeDownload slide Three leaf primordia are evident at the shoot apical meristem (asterisk). The oldest (P3) and P2 primordia encircle the meristem, whereas the youngest (P1) has just been initiated. Scale bar = 20 μm. Venation pattern in expanded leaves To provide a framework on which to map the trajectory of vascular development in embryonic P1–P3 leaf primordia of S. viridis, venation patterns were first examined in the expanded leaf blade of the first leaf to emerge from the coleoptile (i.e. P3 in Fig. 2). Paradermal (Fig. 3A) and transverse (Fig. 3B) views revealed a venation pattern that is typical of monocots, with the longitudinal midvein (MV) and lateral veins (LV) separated by multiple intermediate veins (IV), that will ultimately be distinguishable as two classes (primary with sclerenchyma connecting to the epidermis, and secondary without sclerenchyma). At the widest point of the leaf the midvein is flanked on both sides by two lateral veins whereas at the leaf tip there is just one lateral vein on each side. Transverse (T) veins connect the longitudinal veins at intervals along the proximo-distal leaf axis (Fig. 3C). Fig. 3. View largeDownload slide The vascular system of the first embryonic leaf of S. viridis. (A) Cleared leaf showing the vascular pattern in longitudinal view with midvein (MV) and lateral veins (LV) indicated. (B) Transverse section at the leaf tip showing midvein flanked by a lateral vein on each side, with intermediate veins (IV) interspersed between the major veins. Three layers of ground tissue (dashed red line) separate the two epidermal layers. (C) Vascular pattern in longitudinal section showing midvein, lateral veins and transverse (T) veins. Scale bar = 2 mm (A), 50 µm (B), 100 µm (C). Fig. 3. View largeDownload slide The vascular system of the first embryonic leaf of S. viridis. (A) Cleared leaf showing the vascular pattern in longitudinal view with midvein (MV) and lateral veins (LV) indicated. (B) Transverse section at the leaf tip showing midvein flanked by a lateral vein on each side, with intermediate veins (IV) interspersed between the major veins. Three layers of ground tissue (dashed red line) separate the two epidermal layers. (C) Vascular pattern in longitudinal section showing midvein, lateral veins and transverse (T) veins. Scale bar = 2 mm (A), 50 µm (B), 100 µm (C). Vascular development during the first 36 h of germination To analyse the developmental trajectory towards the expanded leaf venation pattern, transverse sections of developing P3 primordia were examined at different points along the proximo-distal leaf axis, at each of stages S0 to S4. Data from these sections were used to reconstruct the venation pattern at the level of the whole primordium (Fig. 4). Over the first 15 h, the primordium increased in size by only ~10 % in the medio-lateral direction and ~45 % in the proximo-distal direction, and no new veins were initiated (Fig. 4A–C). A further 33 % size increase in the medio-lateral direction between 15 and 24 h led to the initiation of a new intermediate vein at each leaf margin (Fig. 4D). Between 24 and 36 h the primordium increased in size by 50 % in the medio-lateral axis and ~280 % in the proximo-distal axis, but no new veins were initiated (Fig. 4E). Fig. 4. View largeDownload slide Vascular pattern in longitudinal view from midvein to margin on one side of S. viridis leaf from stage S0 (dry seed) to S4 (36 h after imbibition). (A) S0: ~250 × ~330 µm; (B) S1: ~330 × ~365 µm; (C) S2: 360 × ~375 µm; (D) S3: ~980 × ~500 µm; (E) S4: ~2740 × ~725 µm. Numbers in each case refer to length along proximo-distal axis × width of half of medio-lateral axis. Arrows indicate the direction of vein formation – acropetal: midvein (black arrow) and lateral veins (red arrows); basipetal: intermediate veins (black dashed arrows). Scale bar = 125 µm. Fig. 4. View largeDownload slide Vascular pattern in longitudinal view from midvein to margin on one side of S. viridis leaf from stage S0 (dry seed) to S4 (36 h after imbibition). (A) S0: ~250 × ~330 µm; (B) S1: ~330 × ~365 µm; (C) S2: 360 × ~375 µm; (D) S3: ~980 × ~500 µm; (E) S4: ~2740 × ~725 µm. Numbers in each case refer to length along proximo-distal axis × width of half of medio-lateral axis. Arrows indicate the direction of vein formation – acropetal: midvein (black arrow) and lateral veins (red arrows); basipetal: intermediate veins (black dashed arrows). Scale bar = 125 µm. Differentiation of vascular bundles At the tip of the P3 primordium the midvein was flanked by a single lateral vein on each side at all stages of development. In the dry seed (S0), the P3 leaf tip comprised the ground meristem sandwiched by the abaxial and adaxial protoderm. Three procambial centres were identified as groups of small cells but there was no evidence of differentiated cell types at this stage (Fig. 5A). Cells in the procambial centre were more organized at S1 and the intervening ground tissue comprised three layers (Fig. 5B). Protoxylem was evident in the midvein at S2 (Fig. 5C), protophloem at S3 (Fig. 5D) and vacuolated BS cells at S4 (Fig. 5E). By S4, most leaf tissues were differentiated: epidermal cells were expanded; stomata were developed on both surfaces; two to three mesophyll cells were present between each pair of vascular bundles; protoxylem, metaxylem and phloem were all present in the midvein; chloroplasts were visible in both bundle sheath and mesophyll cells; and sclerenchyma was present at the leaf margins (Fig. 5E). Fig. 5. View largeDownload slide Transverse sections at the tip of the P3 primordium. (A) Stage S0 (dry seed) at ~40 µm from the tip. MV – black dashed line and LV – red dashed line. (B–E) Sections at ~60 µm from the tip: Stage S1 (12 h after imbibition) (B); Stage S2 (15 h after imbibition), black arrow indicates protoxylem (C); Stage S3 (24 h after imbibition), yellow arrow indicates protophloem (D); Stage S4 (36 h after imbibition) * = BS cell; ** = M cell; red asterisk = metaxylem; red arrow points to terminating intermediate vein (E). All sections orientated with adaxial surface facing upwards. Scale bar = 20 µm. Fig. 5. View largeDownload slide Transverse sections at the tip of the P3 primordium. (A) Stage S0 (dry seed) at ~40 µm from the tip. MV – black dashed line and LV – red dashed line. (B–E) Sections at ~60 µm from the tip: Stage S1 (12 h after imbibition) (B); Stage S2 (15 h after imbibition), black arrow indicates protoxylem (C); Stage S3 (24 h after imbibition), yellow arrow indicates protophloem (D); Stage S4 (36 h after imbibition) * = BS cell; ** = M cell; red asterisk = metaxylem; red arrow points to terminating intermediate vein (E). All sections orientated with adaxial surface facing upwards. Scale bar = 20 µm. At the widest part of the P3 primordium, 25 procambial centres were observed up to S2 (the midvein, four lateral veins and 20 intermediate veins) (Figs 4A, C and 6A–C), and 27 at S3 and S4 (two more intermediate veins having developed, one at each leaf margin) (Figs 4D, E and 6D, E). A comparison of midvein anatomy at the middle and at the tip of the leaf revealed the normal basipetal differentiation gradient seen in grass leaves. Midvein development was at an equivalent stage 24 h after imbibition at the tip and 36 h after imbibition in the middle of the leaf (compare Figs 5D and 6G). The transition from undifferentiated procambium at the midvein (Fig. 6F) to visible protoxylem and protophloem (Fig. 6G) took place over 24 h. Fig. 6. View largeDownload slide Transverse sections at the widest part of the P3 primordium. (A) Stage S0 (dry seed) at ~180 µm from the tip. (B) Stage S1 (12 h after imbibition) at ~230 µm from the tip. (C) Stage S2 (15 h after imbibition) at ~240 µm from the tip. (D) Stage S3 (24 h after imbibition) at ~690 µm from the tip. (E) Stage S4 (36 h after imbibition) at ~1900 µm from the tip. (F) High magnification of (B) showing the midvein. (G) High magnification of (E) showing the midvein. (A–E) P3 primordium and midvein – dashed black line, P2 primordium – dashed red line, shoot apical meristem – asterisk. (F, G) Bundle sheath progenitor cells dashed black. Scale bar = 50 µm (A–E), 20 µm (F, G). Fig. 6. View largeDownload slide Transverse sections at the widest part of the P3 primordium. (A) Stage S0 (dry seed) at ~180 µm from the tip. (B) Stage S1 (12 h after imbibition) at ~230 µm from the tip. (C) Stage S2 (15 h after imbibition) at ~240 µm from the tip. (D) Stage S3 (24 h after imbibition) at ~690 µm from the tip. (E) Stage S4 (36 h after imbibition) at ~1900 µm from the tip. (F) High magnification of (B) showing the midvein. (G) High magnification of (E) showing the midvein. (A–E) P3 primordium and midvein – dashed black line, P2 primordium – dashed red line, shoot apical meristem – asterisk. (F, G) Bundle sheath progenitor cells dashed black. Scale bar = 50 µm (A–E), 20 µm (F, G). Ultrastructure of developing leaf vascular centres To determine the ultrastructure of developing vascular centres, TEM was carried out on the tip/middle portion of P3 primordia. TEM revealed organized clusters of essentially undifferentiated cells in the dry seed (S0) (Fig. 7A). BS cells could be distinguished by their concentric organization around the procambial cells and by their larger size. Neither protoxylem nor protophloem was differentiated at this point. All cells in these rudimentary vascular centres possessed numerous lipid bodies and the nuclei featured a prominent nucleolus (Fig. 7A). The presence of large amounts of lipid was confirmed by histochemical analysis (Sudan III and Sudan IV) (data not shown). By S1, the number of lipid bodies had decreased and cells in the procambial centre were more organized (Fig. 7B). Initiation of protoxylem development was marked by cell-wall thickening at S2 (Fig. 7C), which was even more evident at S3 (Fig. 7D). At S4, protoxylem, protophloem and metaxylem with degenerated cytoplasm and thick cell walls were evident in the vascular tissue (Fig. 7E). Fig. 7. View largeDownload slide Transmission electron micrographs of transverse sections of developing major veins at the tip of P3 primordia. (A, B) Sections at S0 (dry seed) (A) and S1 (B) showing procambial centres (red dashed line) surrounded by BS progenitor cells (dashed yellow line). Cells contain numerous lipid bodies (orange asterisk), dense cytoplasm and prominent nucleoli (white asterisk). At S1, many lipid bodies have coalesced. (C) S2 section showing protoxylem cells in the procambial centre with thickened cell walls (blue asterisks). Lipid content in all cells is greatly reduced relative to S0 and S1. (D) S3 section showing BS cells with dense cytoplasm, large nuclei (N) and numerous chloroplasts with prominent thylakoid membranes (c). In the vascular centre, progressive thickening of xylem cell walls is evident (red asterisks) and protophloem is in the early stage of development (yellow asterisks). (E) S4 section showing differentiated procambial and BS cells. Protophloem (black asterisks), protoxylem and metaxylem (X) are evident in the vascular centre. BS cells are vacuolated (V) with numerous well-developed chloroplasts (C) that contain starch granules (yellow arrow). CC = companion cells. Scale bar = 5 µm (A–E). Fig. 7. View largeDownload slide Transmission electron micrographs of transverse sections of developing major veins at the tip of P3 primordia. (A, B) Sections at S0 (dry seed) (A) and S1 (B) showing procambial centres (red dashed line) surrounded by BS progenitor cells (dashed yellow line). Cells contain numerous lipid bodies (orange asterisk), dense cytoplasm and prominent nucleoli (white asterisk). At S1, many lipid bodies have coalesced. (C) S2 section showing protoxylem cells in the procambial centre with thickened cell walls (blue asterisks). Lipid content in all cells is greatly reduced relative to S0 and S1. (D) S3 section showing BS cells with dense cytoplasm, large nuclei (N) and numerous chloroplasts with prominent thylakoid membranes (c). In the vascular centre, progressive thickening of xylem cell walls is evident (red asterisks) and protophloem is in the early stage of development (yellow asterisks). (E) S4 section showing differentiated procambial and BS cells. Protophloem (black asterisks), protoxylem and metaxylem (X) are evident in the vascular centre. BS cells are vacuolated (V) with numerous well-developed chloroplasts (C) that contain starch granules (yellow arrow). CC = companion cells. Scale bar = 5 µm (A–E). At S3, BS cells surrounding the developing vein exhibited dense cytoplasm, large nuclei and numerous plastids distributed around the cell periphery (Fig. 7D). By S4, BS cells were vacuolated and chloroplasts contained multiple starch granules (Fig. 7E). BS chloroplasts were qualitatively similar to M chloroplasts in terms of thylakoid stacking at S3 (Fig. 8A) but at S4 M chloroplasts appeared to contain more grana (Fig. 8B, C). Plasmodesmatal connections between BS and M cells were established by S4 (Fig. 8C). Fig. 8. View largeDownload slide Transmission electron micrographs of transverse sections of chloroplasts at the tip of P3 primordia. (A) S3 section showing adjacent M, BS and procambium (Pc) cells. At this stage, some cells are vacuolated whilst others are densely cytoplasmic, and M cells can still contain numerous lipid bodies. Both BS and M cells contain small chloroplasts with minimal thylakoid stacking (black arrows). (B, C) Sections at S4 showing BS (B) and M (C) chloroplasts in vacuolated cells. Areas of stacked thylakoids are visible in chloroplasts of both cell types (yellow arrows). Pc cells contain degenerating protoplasm. Plasmodesmatal connections have formed in the cell walls between BS and M cells (red asterisk). Scale bar = 2 µm (A), 1 µm (B, C). Fig. 8. View largeDownload slide Transmission electron micrographs of transverse sections of chloroplasts at the tip of P3 primordia. (A) S3 section showing adjacent M, BS and procambium (Pc) cells. At this stage, some cells are vacuolated whilst others are densely cytoplasmic, and M cells can still contain numerous lipid bodies. Both BS and M cells contain small chloroplasts with minimal thylakoid stacking (black arrows). (B, C) Sections at S4 showing BS (B) and M (C) chloroplasts in vacuolated cells. Areas of stacked thylakoids are visible in chloroplasts of both cell types (yellow arrows). Pc cells contain degenerating protoplasm. Plasmodesmatal connections have formed in the cell walls between BS and M cells (red asterisk). Scale bar = 2 µm (A), 1 µm (B, C). DISCUSSION The development of S. viridis, from germination to coleoptile rupture 36 h after imbibition, has been investigated using optical, scanning and transmission electron microscopy. Examination of dry seeds demonstrated that three leaves are initiated during embryo development with P3 and P2 primordia encircling the SAM of the mature embryo and P1 comprising just a few cells. By 36 h after imbibition of dry seeds it was possible to identify (1) the NADP-ME ‘Classic’ subtype of Kranz anatomy with large agranal chloroplasts assuming a centrifugal position in the BS cells and no mestome sheath; (2) vacuolated BS and M cells; (3) two to three M cells between adjacent vascular bundles; (4) a single M cell layer between minor veins and the epidermis; and (5) protophloem, protoxylem and metaxylem in developing veins. During the first 12 h after seed imbibition, conspicuous and numerous lipid bodies were present in cells of the embryonic S. viridis leaves (Fig. 7A, B) but by 24 h most of the lipid reserve had been mobilized (Fig. 7D). This form of energy storage has previously been reported in S. lutescens, which showed a decrease in lipid content before leaf emergence after hydration of non-dormant embryos (Rost, 1972), and a similar mobilization of seed reserves precedes cell type differentiation in maize (Nikiforidis et al., 2013). Distinct growth phases are also seen in young leaves of the NADP-ME grass Stenotaphrum secundatum (Sud and Dengler, 2000) and in Cleome angustifolia, an NAD-ME eudicot species (Koteyeva et al., 2014). During the first growth phase in C. angustifolia the meristematic zone of the leaf remains undifferentiated, with BS and M cells only becoming identifiable in the second period of growth. The third phase is characterized by BS cells with well-developed central vacuoles and, during the fourth phase, the characteristic NAD-ME-type differentiation of chloroplasts and mitochondria takes place (Koteyeva et al., 2014). Events in these growth phases in C. angustifolia clearly parallel development during stages S0 to S4 in S. viridis (Figs 5–7). It thus appears that cell type differentiation in embryonic leaves is generally co-ordinated with regulation of the glyoxylate cycle such that reserves are appropriately mobilized prior to intracellular organization. Whereas most previous reports have focused on vascular development in the seedlings of C4 plants, this study has focused on development in embryonic leaves. In grasses the number of embryonic leaves in the seed and the number of juvenile leaves in the seedling are not necessarily the same (Sylvester et al., 2001), and the developmental events determining this relationship are not understood (Bongard-Pierce et al., 1996; Sylvester et al., 2001). For example, in maize the four to five embryonic leaves in the seed correspond to the juvenile leaves in the seedling (Bongard-Pierce et al., 1996, Liu et al., 2013) whereas in bluegrass and rice there are two embryonic leaves but no juvenile seedling leaves (Sylvester et al., 2001). Setaria viridis has four juvenile seedling leaves (Hodge and Doust, 2017) but only three leaves are initiated during embryogenesis, and only two of those exhibit any cell type patterning in the mature embryo (Figs 2 and 6). As such, the signals that pattern vascular development in S. viridis operate in at least three different contexts: (1) juvenile patterned during embryogenesis (first two leaves to emerge), (2) juvenile patterned post-germination (leaves 3 and 4) and (3) adult. Vascular development in embryonic leaves of S. viridis closely resembles that of maize (Liu et al., 2013) and of Spartina alternifolia (Walsh, 1990), with leaf primordia of the mature seed exhibiting several vascular centres at different stages of development (Figs 5 and 6). When the embryo of S. viridis initiated growth following imbibition, the midvein and lateral veins differentiated more rapidly at the tip of the leaf primordium, suggesting that vascular differentiation occurred basipetally, despite the veins being initiated at the base. This same pattern is observed in maize and other grasses (Sharman, 1942; Bosabalidis et al., 1994; Nelson and Dengler, 1997). Studies in maize have suggested that the differentiation of BS and M cells in C4 leaves is dependent on inductive signals from developing veins (Langdale et al., 1988, 1989; Langdale and Nelson, 1991). BS cell development has also been proposed to act as a point of reference, guiding the differentiation of adjacent tissues (Nelson and Dengler, 1997). For example, the position of veins has been shown to play a critical role in the development of the epidermis in maize (Cerioli, et al., 1994). In S. viridis, the consecutive initiation of vascular, BS and epidermal cell types (Figs 5 and 7) supports the view that the formation of vascular bundles influences the organization of adjacent tissues. The development of the C4 photosynthetic apparatus requires co-ordinated gene expression to underpin both anatomical and metabolic traits. Transcriptomes of BS and M cells in maize and S. viridis show a high correlation of transcripts encoding C4 metabolism proteins (John et al., 2014) but similar datasets are not yet available for genes that are likely to regulate leaf anatomy. The anatomical and ultrastructural analyses reported here provide an empirical basis for such comparisons in the future. ACKNOWLEDGEMENTS We thank the Núcleo Multidisciplinar de Pesquisa (NUMPEX Bio, UFRJ) for the use of microscopes and ultramicrotome, the Instituto de Biofisica Carlos Chagas Filho (IBCCF-UFRJ) for the preparation of samples for electron microscopy, and Prof. Marcos Farina of Instituto de Ciências Biomédicas (ICB-UFRJ) for the use of microscopes. We are grateful to the Conselho Nacional de Desenvolvimento Cientıífico e Tecnológico – CNPq for a PhD grant to N.E.G.J. Research was supported in part by a Royal Society Newton Advanced Fellowship to M.A.F. and J.A.L. LITERATURE CITED Aliscioni S , Ospina JC , Gomiz NE . 2016 . 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Leaf vascular pattern formation . The Plant Cell 9 : 1121 – 1135 . Google Scholar CrossRef Search ADS PubMed Nikiforidis CV , Kiosseoglou V , Scholten E . 2013 . Oil bodies: An insight on their microstructure – maize germ vs sunflower seed . Food Research International 52 : 136 – 141 . Google Scholar CrossRef Search ADS Rost TL . 1972 . The ultrastructure and physiology of protein bodies and lipids from hydrated dormant and nondormant embryos of Setaria lutescens (Gramineae) . Botanical Society of America 59 : 607 – 616 . Sage RF . 2002 . C4 photosynthesis in terrestrial plants does not require Kranz anatomy . Trends in Plant Science 7 : 283 – 285 . Google Scholar CrossRef Search ADS PubMed Sage RF , Christin PA , Edwards EJ . 2011 . The C4 plant lineages of planet Earth . Journal of Experimental Botany 62 : 3155 – 3169 . Google Scholar CrossRef Search ADS PubMed Saha P , Blumwald E . 2016 . Spike-dip transformation of Setaria viridis . Plant Journal 86 : 89 – 101 . 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Anatomy and ultrastructure of embryonic leaves of the C4 species Setaria viridis

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
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10.1093/aob/mcx217
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Abstract

Abstract Background and Aims Setaria viridis is being promoted as a model C4 photosynthetic plant because it has a small genome (~515 Mb), a short life cycle (~60 d) and it can be transformed. Unlike other C4 grasses such as maize, however, there is very little information about how C4 leaf anatomy (Kranz anatomy) develops in S. viridis. As a foundation for future developmental genetic studies, we provide an anatomical and ultrastructural framework of early shoot development in S. viridis, focusing on the initiation of Kranz anatomy in seed leaves. Methods Setaria viridis seeds were germinated and divided into five stages covering development from the dry seed (stage S0) to 36 h after germination (stage S4). Material at each of these stages was examined using conventional light, scanning and transmission electron microscopy. Key Results Dry seeds contained three embryonic leaf primordia at different developmental stages (plastochron 1–3 primordia). The oldest (P3) leaf primordium possessed several procambial centres whereas P2 displayed only ground meristem. At the tip of P3 primordia at stage S4, C4 leaf anatomy typical of the malate dehydrogenase-dependent nicotinamide dinucleotide phosphate (NADP-ME) subtype was evident in that vascular bundles lacked a mestome layer and were surrounded by a single layer of bundle sheath cells that contained large, centrifugally located chloroplasts. Two to three mesophyll cells separated adjacent vascular bundles and one mesophyll cell layer on each of the abaxial and adaxial sides delimited vascular bundles from the epidermis. Conclusions The morphological trajectory reported here provides a foundation for studies of gene regulation during early leaf development in S. viridis and a framework for comparative analyses with other C4 grasses. C4, Setaria viridis, Kranz anatomy, embryonic leaves, vascular development INTRODUCTION C4 photosynthesis occurs in 19 angiosperm families that encompass both monocots and eudicots, with monocot representatives including ~4500 grass species (Sage et al., 2011; Williams et al., 2012). The C4 pathway differs from the C3 pathway in that photosynthesis is split between two cell types. CO2 is initially fixed in the mesophyll (M) cells by phosphoenolpyruvate carboxylase and then after decarboxylation of a C4 acid, it is re-fixed by ribulose bisphosphate carboxylase/oxygenase in the bundle sheath (BS) cells (Von Caemmerer and Furbank, 2003; Langdale, 2011). In most C4 plants, BS and M cell types are arranged around leaf veins in a characteristic anatomy known as Kranz (Brown, 1975; Dengler et al., 1985). Although Kranz anatomy is not a prerequisite for C4 photosynthesis (Voznesenskaya et al., 2001; Sage, 2002), it is present in all C4 grasses. C4 plants have previously been classified into three subtypes, depending on the mechanism by which C4 acids are decarboxylated in the BS cells. Although the biochemical distinction between these subtypes is now questioned (Furbank, 2011), there are distinct differences between BS cell anatomy in so-called malate dehydrogenase-dependent nicotinamide dinucleotide phosphate (NADP-ME), NAD-ME and PEP-CK subtypes (Brown, 1975; Edwards and Voznesenskaya, 2011). In NADP-ME species, BS cell chloroplasts lack well-developed grana and are located centrifugally; in NAD-ME species, BS chloroplasts are positioned centripetally and possess well-developed grana; and in PEP-CK species, BS chloroplasts possess well-developed grana and are arranged in a centrifugal position. In addition to differences in chloroplast structure and arrangement, BS cells are distinguished from M cells by a range of other features including number, arrangement and size of mitochondria, degree of vacuolation and type of plasmodesmata. The thickness and composition of the cell wall can also differ (Dengler et al., 1986; Edwards and Voznesenskaya, 2011). For example, the BS cells of several species in the NADP-ME and PEP-CK subgroups have a suberized lamella (Eastman, 1988; Edwards and Voznesenskaya, 2011). With at least 62 lineages of C4 plants now identified (Sage et al., 2011), further variation in structure and organization of Kranz anatomy is likely to be discovered (Williams et al., 2012), reinforcing the need for a clear and detailed understanding of both anatomy and metabolism in species used as C4 models. The C4 grass Setaria viridis has been proposed as a model for understanding C4 metabolism and anatomy (Brutnell et al., 2010; Diao et al., 2014), with the genome sequence of closely related S. italica (Bennetzen et al., 2012) and transformation protocols (Martins et al., 2015; Van Eck and Swartwood, 2015; Saha and Blumwald, 2016) opening the way for comprehensive analyses of C4 photosynthesis in a rapid cycling plant. Setaria viridis is an NADP-ME C4 grass (Aliscioni et al., 2016), but is phylogenetically related to switchgrass (NAD-ME subtype) and Panicum virgatum (PEP-CK subtype) (Brutnell et al., 2010). Six subclasses of NADP-ME subtype anatomy have been identified in Poaceae, including the most common ‘Classic’ subtype (Edwards and Voznesenskaya, 2011). The different anatomies mainly vary with respect to (1) presence or absence of the mestome sheath and suberized lamella, (2) number of BS cell layers and (3) ultrastructure of chloroplasts (Edwards and Voznesenskaya, 2011). Because photosynthetic BS cells are a defining feature of C4 plants, understanding the ontogeny of this cell type is central to any interpretation of the function of these different anatomical arrangements and of the relationship between species possessing them. We have therefore conducted a detailed analysis of the structure and the ultrastructure of BS cells in embryonic leaves of S. viridis during germination, from seed imbibition to coleoptile rupture by the first leaf. The data presented here on the pattern of Kranz differentiation in S. viridis seed leaves provide a platform for future morphological and comparative studies with this new C4 model species. MATERIALS AND METHODS Plant growth and sampling Fruit of Setaria viridis (L.) Beauvois accession A10.1 were collected from greenhouse-grown plants at the Universidade Federal do Rio de Janeiro - UFRJ (Brazil) in January 2015 and stored at room temperature (25–28 °C) for 10 months before use. Prior to sampling for microscopy, ~150 fruits were placed in 30 mm potassium nitrate solution for 24 h at room temperature and then washed three times with gentle agitation in distilled water (modified from Sebastian et al., 2014). Germination was then carried out on moistened Germitest paper (Germilab, Brazil) in Petri dishes in a BOD incubator (Eletrolab EL202, SP, Brazil) at 28 °C under continuous light. For light and transmission electron microscopy (TEM), ten fruits with bracts were sampled at each of the sampling times: 0 h (dry seed), 12 h, 15 h, 24 h and 36 h after imbibition. Sampling times were established based on pilot studies that identified morphological landmarks such as the protrusion of coleorhiza and the appearance of the primary root. The developmental stage of the seed at each sampling time was classified as S0 (0 h), S1 (12 h), S2 (15 h), S3 (24 h) and S4 (36 h). Seeds became swollen between stages S0 and S1, and the coleorhiza emerged at stage Sl. Stage S2 was characterized by the presence of hairs on the coleorhiza and stage S3 by the appearance of the coleoptile and the primary root. By stage S4, the first embryonic leaf had ruptured the coleoptile. Five fruits without bracts were collected for scanning electron microscopy (SEM) at the same sampling times. All bracts (two glumes, sterile lemma, fertile lemma and palea) were removed from the fruits to facilitate SEM study of post-germination events. To examine the overall venation pattern in expanded leaves, the first leaf to emerge was harvested around 1 week after seed imbibition. The leaf was cleared with ethanol at 90 °C and stained in 1 % safranin (modified from Kraus and Arduin, 1997). Anatomical and ultrastructural studies Plant material was fixed in 4 % paraformaldehyde and 2.5 % glutaraldehyde in 0.1 m phosphate buffer for 24 h and post-fixed in 1 % osmium tetroxide in 0.1 m phosphate buffer for 1 h. For SEM, fruits without bracts were then dehydrated through an ethanol gradient, critical-point dried and sputter-coated with gold (Vega 3LMU Tescan, Brno, Czech Republic). Samples were viewed using a scanning electron microscope (Spirit Biotwin 12, FEI Company, Hillsboro, OR, USA). For light microscopy, fruits with bracts were dehydrated in an ethanol/propylene oxide series, embedded in Spurr’s resin (Premix Kit-Hard, TAAB Laboratory and Microscopy, Aldermaston, UK) and polymerized at 70 °C overnight. Transverse thin sections (1 µm) of the embedded material were cut from the tip to the base of the coleoptile using an ultramicrotome (RMC Products, Tucson, AZ, USA), stained with 0.05 % toluidine blue and viewed in a Leica DM 2500 (Wetzlar, Germany) optical microscope. Sequential 1-µm sections were used to create longitudinal diagrams of the leaf vascular pattern based on the structures present at each developmental stage. For TEM, ultrathin sections (60 nm) were cut using an ultramicrotome (ultra-RMC Products), mounted on copper grids, and contrasted with 1 % uranyl acetate in water for 25 min followed by lead citrate for 3 min. Sections were examined in a transmission electron microscope (Spirit Biotwin 12 FEI Company). Measurements were carried out using the TEM Imaging Platform program. RESULTS General development during the first 36 h of germination SEM images of the diaspore (dispersion unit) of S. viridis revealed a caryopsis consisting of five bracts – two glumes, a sterile and a fertile lemma, plus a palea (Stage S0; Fig. 1A). The coleorhiza ruptured the seed coat after 12 h of imbibition (Stage Sl; Fig. 1B) and after 15 h the absorbent hairs of the coleorhiza were visible (Stage S2; Fig. 1C). Disruption of the caryopsis as a result of emergence of the coleoptile and the radicle occurred 24 h after imbibition (Stage S3; Fig. 1D), and 36 h after imbibition the first juvenile leaf ruptured the coleoptile (Stage S4; Fig. 1E, F). The diaspore is hereafter referred to as the seed. Fig. 1. View largeDownload slide Scanning electron microscopy of S. viridis diaspore during germination from dry seed to 36 h after imbibition (first embryonic leaf rupturing the coleoptile). (A) Stage S0 – dry seed – with bracts: first glume (*), second glume (**), sterile lemma (arrow) and fertile lemma (dashed arrow) enveloping the caryopsis (the palea remains covered by the sterile lemma). (B) Stage S1 – 12 h after imbibition, the coleorhiza rupturing the caryopsis (arrow). (C) Stage S2 – 15 h after imbibition, absorbent hairs of the coleorhiza (arrow) are visible. (D) Stage S3 – 24 h after imbibition, coleoptile breaking through the caryopsis (arrow); the radicle (*), the mesocotyl (dashed arrow) and coleorhiza (**) are visible. (E) Stage S4 – 36 h after imbibition, left: intact coleoptile; right: the first juvenile leaf has ruptured the coleoptile (arrow). (F) Ruptured coleoptile shown in detail. Scale bar = 500 μm (A, B, D), 1mm (C), 2 mm (E), 200 μm (F). Fig. 1. View largeDownload slide Scanning electron microscopy of S. viridis diaspore during germination from dry seed to 36 h after imbibition (first embryonic leaf rupturing the coleoptile). (A) Stage S0 – dry seed – with bracts: first glume (*), second glume (**), sterile lemma (arrow) and fertile lemma (dashed arrow) enveloping the caryopsis (the palea remains covered by the sterile lemma). (B) Stage S1 – 12 h after imbibition, the coleorhiza rupturing the caryopsis (arrow). (C) Stage S2 – 15 h after imbibition, absorbent hairs of the coleorhiza (arrow) are visible. (D) Stage S3 – 24 h after imbibition, coleoptile breaking through the caryopsis (arrow); the radicle (*), the mesocotyl (dashed arrow) and coleorhiza (**) are visible. (E) Stage S4 – 36 h after imbibition, left: intact coleoptile; right: the first juvenile leaf has ruptured the coleoptile (arrow). (F) Ruptured coleoptile shown in detail. Scale bar = 500 μm (A, B, D), 1mm (C), 2 mm (E), 200 μm (F). Anatomy of the mature embryo The embryo of mature seeds averages ~800 µm in length and ~150 µm in width, excluding the coleoptile. The shoot apical meristem (SAM) and young leaf primordia are subtended by the mesocotyl, which is characterized by cells that are more densely cytoplasmic than in the SAM, and by the radicle in which longitudinal cell files are apparent. Three leaf primordia are present around the SAM (Fig. 2), referred to as plastochrons (P) 1–3, where a plastochron is the time interval between initiation of sequential primordia. The youngest (P1) primordium has just initiated whereas the P2 and P3 primordia enclose the SAM, as is typical of grass leaf primordia. During the period 0–36 h examined here, no new leaf primordia were initiated at the SAM. Fig. 2. View largeDownload slide Three leaf primordia are evident at the shoot apical meristem (asterisk). The oldest (P3) and P2 primordia encircle the meristem, whereas the youngest (P1) has just been initiated. Scale bar = 20 μm. Fig. 2. View largeDownload slide Three leaf primordia are evident at the shoot apical meristem (asterisk). The oldest (P3) and P2 primordia encircle the meristem, whereas the youngest (P1) has just been initiated. Scale bar = 20 μm. Venation pattern in expanded leaves To provide a framework on which to map the trajectory of vascular development in embryonic P1–P3 leaf primordia of S. viridis, venation patterns were first examined in the expanded leaf blade of the first leaf to emerge from the coleoptile (i.e. P3 in Fig. 2). Paradermal (Fig. 3A) and transverse (Fig. 3B) views revealed a venation pattern that is typical of monocots, with the longitudinal midvein (MV) and lateral veins (LV) separated by multiple intermediate veins (IV), that will ultimately be distinguishable as two classes (primary with sclerenchyma connecting to the epidermis, and secondary without sclerenchyma). At the widest point of the leaf the midvein is flanked on both sides by two lateral veins whereas at the leaf tip there is just one lateral vein on each side. Transverse (T) veins connect the longitudinal veins at intervals along the proximo-distal leaf axis (Fig. 3C). Fig. 3. View largeDownload slide The vascular system of the first embryonic leaf of S. viridis. (A) Cleared leaf showing the vascular pattern in longitudinal view with midvein (MV) and lateral veins (LV) indicated. (B) Transverse section at the leaf tip showing midvein flanked by a lateral vein on each side, with intermediate veins (IV) interspersed between the major veins. Three layers of ground tissue (dashed red line) separate the two epidermal layers. (C) Vascular pattern in longitudinal section showing midvein, lateral veins and transverse (T) veins. Scale bar = 2 mm (A), 50 µm (B), 100 µm (C). Fig. 3. View largeDownload slide The vascular system of the first embryonic leaf of S. viridis. (A) Cleared leaf showing the vascular pattern in longitudinal view with midvein (MV) and lateral veins (LV) indicated. (B) Transverse section at the leaf tip showing midvein flanked by a lateral vein on each side, with intermediate veins (IV) interspersed between the major veins. Three layers of ground tissue (dashed red line) separate the two epidermal layers. (C) Vascular pattern in longitudinal section showing midvein, lateral veins and transverse (T) veins. Scale bar = 2 mm (A), 50 µm (B), 100 µm (C). Vascular development during the first 36 h of germination To analyse the developmental trajectory towards the expanded leaf venation pattern, transverse sections of developing P3 primordia were examined at different points along the proximo-distal leaf axis, at each of stages S0 to S4. Data from these sections were used to reconstruct the venation pattern at the level of the whole primordium (Fig. 4). Over the first 15 h, the primordium increased in size by only ~10 % in the medio-lateral direction and ~45 % in the proximo-distal direction, and no new veins were initiated (Fig. 4A–C). A further 33 % size increase in the medio-lateral direction between 15 and 24 h led to the initiation of a new intermediate vein at each leaf margin (Fig. 4D). Between 24 and 36 h the primordium increased in size by 50 % in the medio-lateral axis and ~280 % in the proximo-distal axis, but no new veins were initiated (Fig. 4E). Fig. 4. View largeDownload slide Vascular pattern in longitudinal view from midvein to margin on one side of S. viridis leaf from stage S0 (dry seed) to S4 (36 h after imbibition). (A) S0: ~250 × ~330 µm; (B) S1: ~330 × ~365 µm; (C) S2: 360 × ~375 µm; (D) S3: ~980 × ~500 µm; (E) S4: ~2740 × ~725 µm. Numbers in each case refer to length along proximo-distal axis × width of half of medio-lateral axis. Arrows indicate the direction of vein formation – acropetal: midvein (black arrow) and lateral veins (red arrows); basipetal: intermediate veins (black dashed arrows). Scale bar = 125 µm. Fig. 4. View largeDownload slide Vascular pattern in longitudinal view from midvein to margin on one side of S. viridis leaf from stage S0 (dry seed) to S4 (36 h after imbibition). (A) S0: ~250 × ~330 µm; (B) S1: ~330 × ~365 µm; (C) S2: 360 × ~375 µm; (D) S3: ~980 × ~500 µm; (E) S4: ~2740 × ~725 µm. Numbers in each case refer to length along proximo-distal axis × width of half of medio-lateral axis. Arrows indicate the direction of vein formation – acropetal: midvein (black arrow) and lateral veins (red arrows); basipetal: intermediate veins (black dashed arrows). Scale bar = 125 µm. Differentiation of vascular bundles At the tip of the P3 primordium the midvein was flanked by a single lateral vein on each side at all stages of development. In the dry seed (S0), the P3 leaf tip comprised the ground meristem sandwiched by the abaxial and adaxial protoderm. Three procambial centres were identified as groups of small cells but there was no evidence of differentiated cell types at this stage (Fig. 5A). Cells in the procambial centre were more organized at S1 and the intervening ground tissue comprised three layers (Fig. 5B). Protoxylem was evident in the midvein at S2 (Fig. 5C), protophloem at S3 (Fig. 5D) and vacuolated BS cells at S4 (Fig. 5E). By S4, most leaf tissues were differentiated: epidermal cells were expanded; stomata were developed on both surfaces; two to three mesophyll cells were present between each pair of vascular bundles; protoxylem, metaxylem and phloem were all present in the midvein; chloroplasts were visible in both bundle sheath and mesophyll cells; and sclerenchyma was present at the leaf margins (Fig. 5E). Fig. 5. View largeDownload slide Transverse sections at the tip of the P3 primordium. (A) Stage S0 (dry seed) at ~40 µm from the tip. MV – black dashed line and LV – red dashed line. (B–E) Sections at ~60 µm from the tip: Stage S1 (12 h after imbibition) (B); Stage S2 (15 h after imbibition), black arrow indicates protoxylem (C); Stage S3 (24 h after imbibition), yellow arrow indicates protophloem (D); Stage S4 (36 h after imbibition) * = BS cell; ** = M cell; red asterisk = metaxylem; red arrow points to terminating intermediate vein (E). All sections orientated with adaxial surface facing upwards. Scale bar = 20 µm. Fig. 5. View largeDownload slide Transverse sections at the tip of the P3 primordium. (A) Stage S0 (dry seed) at ~40 µm from the tip. MV – black dashed line and LV – red dashed line. (B–E) Sections at ~60 µm from the tip: Stage S1 (12 h after imbibition) (B); Stage S2 (15 h after imbibition), black arrow indicates protoxylem (C); Stage S3 (24 h after imbibition), yellow arrow indicates protophloem (D); Stage S4 (36 h after imbibition) * = BS cell; ** = M cell; red asterisk = metaxylem; red arrow points to terminating intermediate vein (E). All sections orientated with adaxial surface facing upwards. Scale bar = 20 µm. At the widest part of the P3 primordium, 25 procambial centres were observed up to S2 (the midvein, four lateral veins and 20 intermediate veins) (Figs 4A, C and 6A–C), and 27 at S3 and S4 (two more intermediate veins having developed, one at each leaf margin) (Figs 4D, E and 6D, E). A comparison of midvein anatomy at the middle and at the tip of the leaf revealed the normal basipetal differentiation gradient seen in grass leaves. Midvein development was at an equivalent stage 24 h after imbibition at the tip and 36 h after imbibition in the middle of the leaf (compare Figs 5D and 6G). The transition from undifferentiated procambium at the midvein (Fig. 6F) to visible protoxylem and protophloem (Fig. 6G) took place over 24 h. Fig. 6. View largeDownload slide Transverse sections at the widest part of the P3 primordium. (A) Stage S0 (dry seed) at ~180 µm from the tip. (B) Stage S1 (12 h after imbibition) at ~230 µm from the tip. (C) Stage S2 (15 h after imbibition) at ~240 µm from the tip. (D) Stage S3 (24 h after imbibition) at ~690 µm from the tip. (E) Stage S4 (36 h after imbibition) at ~1900 µm from the tip. (F) High magnification of (B) showing the midvein. (G) High magnification of (E) showing the midvein. (A–E) P3 primordium and midvein – dashed black line, P2 primordium – dashed red line, shoot apical meristem – asterisk. (F, G) Bundle sheath progenitor cells dashed black. Scale bar = 50 µm (A–E), 20 µm (F, G). Fig. 6. View largeDownload slide Transverse sections at the widest part of the P3 primordium. (A) Stage S0 (dry seed) at ~180 µm from the tip. (B) Stage S1 (12 h after imbibition) at ~230 µm from the tip. (C) Stage S2 (15 h after imbibition) at ~240 µm from the tip. (D) Stage S3 (24 h after imbibition) at ~690 µm from the tip. (E) Stage S4 (36 h after imbibition) at ~1900 µm from the tip. (F) High magnification of (B) showing the midvein. (G) High magnification of (E) showing the midvein. (A–E) P3 primordium and midvein – dashed black line, P2 primordium – dashed red line, shoot apical meristem – asterisk. (F, G) Bundle sheath progenitor cells dashed black. Scale bar = 50 µm (A–E), 20 µm (F, G). Ultrastructure of developing leaf vascular centres To determine the ultrastructure of developing vascular centres, TEM was carried out on the tip/middle portion of P3 primordia. TEM revealed organized clusters of essentially undifferentiated cells in the dry seed (S0) (Fig. 7A). BS cells could be distinguished by their concentric organization around the procambial cells and by their larger size. Neither protoxylem nor protophloem was differentiated at this point. All cells in these rudimentary vascular centres possessed numerous lipid bodies and the nuclei featured a prominent nucleolus (Fig. 7A). The presence of large amounts of lipid was confirmed by histochemical analysis (Sudan III and Sudan IV) (data not shown). By S1, the number of lipid bodies had decreased and cells in the procambial centre were more organized (Fig. 7B). Initiation of protoxylem development was marked by cell-wall thickening at S2 (Fig. 7C), which was even more evident at S3 (Fig. 7D). At S4, protoxylem, protophloem and metaxylem with degenerated cytoplasm and thick cell walls were evident in the vascular tissue (Fig. 7E). Fig. 7. View largeDownload slide Transmission electron micrographs of transverse sections of developing major veins at the tip of P3 primordia. (A, B) Sections at S0 (dry seed) (A) and S1 (B) showing procambial centres (red dashed line) surrounded by BS progenitor cells (dashed yellow line). Cells contain numerous lipid bodies (orange asterisk), dense cytoplasm and prominent nucleoli (white asterisk). At S1, many lipid bodies have coalesced. (C) S2 section showing protoxylem cells in the procambial centre with thickened cell walls (blue asterisks). Lipid content in all cells is greatly reduced relative to S0 and S1. (D) S3 section showing BS cells with dense cytoplasm, large nuclei (N) and numerous chloroplasts with prominent thylakoid membranes (c). In the vascular centre, progressive thickening of xylem cell walls is evident (red asterisks) and protophloem is in the early stage of development (yellow asterisks). (E) S4 section showing differentiated procambial and BS cells. Protophloem (black asterisks), protoxylem and metaxylem (X) are evident in the vascular centre. BS cells are vacuolated (V) with numerous well-developed chloroplasts (C) that contain starch granules (yellow arrow). CC = companion cells. Scale bar = 5 µm (A–E). Fig. 7. View largeDownload slide Transmission electron micrographs of transverse sections of developing major veins at the tip of P3 primordia. (A, B) Sections at S0 (dry seed) (A) and S1 (B) showing procambial centres (red dashed line) surrounded by BS progenitor cells (dashed yellow line). Cells contain numerous lipid bodies (orange asterisk), dense cytoplasm and prominent nucleoli (white asterisk). At S1, many lipid bodies have coalesced. (C) S2 section showing protoxylem cells in the procambial centre with thickened cell walls (blue asterisks). Lipid content in all cells is greatly reduced relative to S0 and S1. (D) S3 section showing BS cells with dense cytoplasm, large nuclei (N) and numerous chloroplasts with prominent thylakoid membranes (c). In the vascular centre, progressive thickening of xylem cell walls is evident (red asterisks) and protophloem is in the early stage of development (yellow asterisks). (E) S4 section showing differentiated procambial and BS cells. Protophloem (black asterisks), protoxylem and metaxylem (X) are evident in the vascular centre. BS cells are vacuolated (V) with numerous well-developed chloroplasts (C) that contain starch granules (yellow arrow). CC = companion cells. Scale bar = 5 µm (A–E). At S3, BS cells surrounding the developing vein exhibited dense cytoplasm, large nuclei and numerous plastids distributed around the cell periphery (Fig. 7D). By S4, BS cells were vacuolated and chloroplasts contained multiple starch granules (Fig. 7E). BS chloroplasts were qualitatively similar to M chloroplasts in terms of thylakoid stacking at S3 (Fig. 8A) but at S4 M chloroplasts appeared to contain more grana (Fig. 8B, C). Plasmodesmatal connections between BS and M cells were established by S4 (Fig. 8C). Fig. 8. View largeDownload slide Transmission electron micrographs of transverse sections of chloroplasts at the tip of P3 primordia. (A) S3 section showing adjacent M, BS and procambium (Pc) cells. At this stage, some cells are vacuolated whilst others are densely cytoplasmic, and M cells can still contain numerous lipid bodies. Both BS and M cells contain small chloroplasts with minimal thylakoid stacking (black arrows). (B, C) Sections at S4 showing BS (B) and M (C) chloroplasts in vacuolated cells. Areas of stacked thylakoids are visible in chloroplasts of both cell types (yellow arrows). Pc cells contain degenerating protoplasm. Plasmodesmatal connections have formed in the cell walls between BS and M cells (red asterisk). Scale bar = 2 µm (A), 1 µm (B, C). Fig. 8. View largeDownload slide Transmission electron micrographs of transverse sections of chloroplasts at the tip of P3 primordia. (A) S3 section showing adjacent M, BS and procambium (Pc) cells. At this stage, some cells are vacuolated whilst others are densely cytoplasmic, and M cells can still contain numerous lipid bodies. Both BS and M cells contain small chloroplasts with minimal thylakoid stacking (black arrows). (B, C) Sections at S4 showing BS (B) and M (C) chloroplasts in vacuolated cells. Areas of stacked thylakoids are visible in chloroplasts of both cell types (yellow arrows). Pc cells contain degenerating protoplasm. Plasmodesmatal connections have formed in the cell walls between BS and M cells (red asterisk). Scale bar = 2 µm (A), 1 µm (B, C). DISCUSSION The development of S. viridis, from germination to coleoptile rupture 36 h after imbibition, has been investigated using optical, scanning and transmission electron microscopy. Examination of dry seeds demonstrated that three leaves are initiated during embryo development with P3 and P2 primordia encircling the SAM of the mature embryo and P1 comprising just a few cells. By 36 h after imbibition of dry seeds it was possible to identify (1) the NADP-ME ‘Classic’ subtype of Kranz anatomy with large agranal chloroplasts assuming a centrifugal position in the BS cells and no mestome sheath; (2) vacuolated BS and M cells; (3) two to three M cells between adjacent vascular bundles; (4) a single M cell layer between minor veins and the epidermis; and (5) protophloem, protoxylem and metaxylem in developing veins. During the first 12 h after seed imbibition, conspicuous and numerous lipid bodies were present in cells of the embryonic S. viridis leaves (Fig. 7A, B) but by 24 h most of the lipid reserve had been mobilized (Fig. 7D). This form of energy storage has previously been reported in S. lutescens, which showed a decrease in lipid content before leaf emergence after hydration of non-dormant embryos (Rost, 1972), and a similar mobilization of seed reserves precedes cell type differentiation in maize (Nikiforidis et al., 2013). Distinct growth phases are also seen in young leaves of the NADP-ME grass Stenotaphrum secundatum (Sud and Dengler, 2000) and in Cleome angustifolia, an NAD-ME eudicot species (Koteyeva et al., 2014). During the first growth phase in C. angustifolia the meristematic zone of the leaf remains undifferentiated, with BS and M cells only becoming identifiable in the second period of growth. The third phase is characterized by BS cells with well-developed central vacuoles and, during the fourth phase, the characteristic NAD-ME-type differentiation of chloroplasts and mitochondria takes place (Koteyeva et al., 2014). Events in these growth phases in C. angustifolia clearly parallel development during stages S0 to S4 in S. viridis (Figs 5–7). It thus appears that cell type differentiation in embryonic leaves is generally co-ordinated with regulation of the glyoxylate cycle such that reserves are appropriately mobilized prior to intracellular organization. Whereas most previous reports have focused on vascular development in the seedlings of C4 plants, this study has focused on development in embryonic leaves. In grasses the number of embryonic leaves in the seed and the number of juvenile leaves in the seedling are not necessarily the same (Sylvester et al., 2001), and the developmental events determining this relationship are not understood (Bongard-Pierce et al., 1996; Sylvester et al., 2001). For example, in maize the four to five embryonic leaves in the seed correspond to the juvenile leaves in the seedling (Bongard-Pierce et al., 1996, Liu et al., 2013) whereas in bluegrass and rice there are two embryonic leaves but no juvenile seedling leaves (Sylvester et al., 2001). Setaria viridis has four juvenile seedling leaves (Hodge and Doust, 2017) but only three leaves are initiated during embryogenesis, and only two of those exhibit any cell type patterning in the mature embryo (Figs 2 and 6). As such, the signals that pattern vascular development in S. viridis operate in at least three different contexts: (1) juvenile patterned during embryogenesis (first two leaves to emerge), (2) juvenile patterned post-germination (leaves 3 and 4) and (3) adult. Vascular development in embryonic leaves of S. viridis closely resembles that of maize (Liu et al., 2013) and of Spartina alternifolia (Walsh, 1990), with leaf primordia of the mature seed exhibiting several vascular centres at different stages of development (Figs 5 and 6). When the embryo of S. viridis initiated growth following imbibition, the midvein and lateral veins differentiated more rapidly at the tip of the leaf primordium, suggesting that vascular differentiation occurred basipetally, despite the veins being initiated at the base. This same pattern is observed in maize and other grasses (Sharman, 1942; Bosabalidis et al., 1994; Nelson and Dengler, 1997). Studies in maize have suggested that the differentiation of BS and M cells in C4 leaves is dependent on inductive signals from developing veins (Langdale et al., 1988, 1989; Langdale and Nelson, 1991). BS cell development has also been proposed to act as a point of reference, guiding the differentiation of adjacent tissues (Nelson and Dengler, 1997). For example, the position of veins has been shown to play a critical role in the development of the epidermis in maize (Cerioli, et al., 1994). In S. viridis, the consecutive initiation of vascular, BS and epidermal cell types (Figs 5 and 7) supports the view that the formation of vascular bundles influences the organization of adjacent tissues. The development of the C4 photosynthetic apparatus requires co-ordinated gene expression to underpin both anatomical and metabolic traits. Transcriptomes of BS and M cells in maize and S. viridis show a high correlation of transcripts encoding C4 metabolism proteins (John et al., 2014) but similar datasets are not yet available for genes that are likely to regulate leaf anatomy. The anatomical and ultrastructural analyses reported here provide an empirical basis for such comparisons in the future. ACKNOWLEDGEMENTS We thank the Núcleo Multidisciplinar de Pesquisa (NUMPEX Bio, UFRJ) for the use of microscopes and ultramicrotome, the Instituto de Biofisica Carlos Chagas Filho (IBCCF-UFRJ) for the preparation of samples for electron microscopy, and Prof. Marcos Farina of Instituto de Ciências Biomédicas (ICB-UFRJ) for the use of microscopes. We are grateful to the Conselho Nacional de Desenvolvimento Cientıífico e Tecnológico – CNPq for a PhD grant to N.E.G.J. Research was supported in part by a Royal Society Newton Advanced Fellowship to M.A.F. and J.A.L. LITERATURE CITED Aliscioni S , Ospina JC , Gomiz NE . 2016 . 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Annals of BotanyOxford University Press

Published: Feb 5, 2018

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