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Figures
- Fig. 1.
Anatomy of wild-type and ral1 mature embryos and plants. (A-E,K,M-P,U,W) Wild type. (F-J,L,Q-T,V,X) ral1. (A-C,F-H) Longitudinal sections through a mature embryo showing details of the embryonic axis (A,F), the shoot apical region (B,G) and the basal region of the scutellum (C,H). Note that the absence of vasculature in F and G, as compared with A and B, is due to the fact that in ral1 the plumule lies slightly off the median plane. (D,I) Detail of the dorsal region of the scutellum in a transverse section through a mature embryo 180 μm below the scutellum tip. (E,J) Schematic representation of a median longitudinal section through a mature embryo (left) and of a dorsal view of a mature embryo (right). The embryonic provascular system is shown in green. (K,L) Region between two large veins in a transverse section through the middle of a mature leaf blade. (M,Q) Midrib region in a transverse section through the middle of a mature leaf blade. Arrows in M indicate the veins of the wild type that are missing in ral1. (N,R) Detail of the upper right-hand corner in M and Q, respectively. (O,S) Detail of the large vascular bundle of the midrib in M and Q, respectively. (P,T) Detail of transverse sections through the apical region of the first internode showing details of the internode wall. (U,V) Transverse section through an adventitious root 12 mm from the root tip. (W,X) Detail of the vascular cylinder in U and V, respectively. Toluidine Blue-stained granules in F,G,I showed typical blue-brown staining with iodine solution (not shown), revealing ectopic starch formation. bs, bundle sheath extension; lv, large vein; mp, metaphloem; mx, late metaxylem element; p, plumule; r, radicle; s, scutellum; sc, sclerenchyma; sv, small vein; v, provascular tissue. Scale bars: (A,F) 150 μm (B,D,G,I,K,L,N,O,R,S) 50 μm (C,H,M,Q,P,T,U,V) 100 μm (W,X) 25 μm.
- Fig. 2.
Morphology of wild-type and ral1 seedlings and mature plants. (A-F) Wild type. (G-L) ral1. (A,G) 3-day-old seedling. (B,H) 6-day-old seedling. (C,I) Root system of a 3-week-old seedling. (D,J) 6-month-old plant. (E,K) Mature spikelet. (F,L) Floral organs in a bisected spikelet. a, adventitious root; l, lemma; p, palea; r, radicle (seminal root). Corresponding ral1 and wild-type images are at the same magnification.
- Table 1.
Morphometric analysis of wild-type and ral1 plants
Wild type Seminal (root) Adventitious (root) ral1 Root Cortical cell size — radial (μm) 28.4±0.8 (40) 23.0±0.3 (107) 31.8±0.5 (101)*** Cortical cell size — tangential (μm) 33.2±1.0 (39) 26.8±0.5 (104) 37.1±0.6 (97)*** Metaxylem element size — radial (μm) 12.3±0.3 (38) 13.1±0.2 (37) 7.2±0.2 (28)*** Metaxylem element size — tangential (μm) 10.3±0.1 (36) 11.0±0.2 (37) 7.9±0.1 (30)*** Number of cortical cell layers 5.0±0.0 (10) 5.1±0.1 (15) 4.0±0.0 (20)*** Number of xylary poles 6.0±0.0 (10) 6.0±0.0 (14) 4.7±0.1 (20)*** Number of adventitious roots 5.2±0.3 (10) 3.0±0.2 (10)*** Number of lateral roots 77.8±6.3 (33) 48.9±5.0 (52) 8.8±2.4 (27)*** Root elongation (mm) 16.0±0.8 (20) 12.1±0.6 (82) 9.8±0.7 (35)*** Wild type ral1 Leaf Blade length (cm) 60.7±1.9 (40) 47.3±2.2 (46)*** Blade width (cm) 1.2±0.0 (40) 0.9±0.0 (46)*** Blade thickness (μm) 49.3±0.9 (48) 53.5±0.7 (71)*** Mesophyll cell size — longitudinal (μm) 9.4±0.1 (123) 9.7±0.1 (105) Mesophyll cell size — radial (μm) 10.2±0.2 (100) 11.3±0.2 (102)*** Mesophyll cell size — tangential (μm) 14.5±0.4 (100) 16.2±0.4 (101)** Metaxylem element size — radial (μm) 66.6±1.8 (24) 43.0±0.5 (24)*** Metaxylem element size — tangential (μm) 52.2±0.7 (24) 41.1±0.3 (24)*** Number of mesophyll cells 3.0±0.1 (45) 3.0±0.0 (57) Number of LVs 9.2±0.2 (40) 7.2±0.2 (46)*** Number of SVs in between two adjacent LVs 5.0±0.4 (20) 3.1±0.1 (38)*** Distance between two adjacent LVs (μm): mature leaves 119.9±2.1 (70) 109.5±1.4 (73)*** immature wt leaves of same length/width as mature ral1 120.7±2.1 (21)/127.7±2.2 (22) 109.3±2.1 (31)**/*** Distance between two adjacent CVs (μm) 618.8±9.8 (66) 760.9±12.8 (74)*** Area enclosed by two CVs and two lVs (μm2) 71866.0±2826.0 (40) 83274.6±2309.9 (48)** Stem Number of vascular bundles in the outer ring 24.9±0.1 (11) 32.1±0.2 (14)*** Number of vascular bundles in the inner ring 13.2±0.1 (11) 14.1±0.1 (14)*** Flower Number of spikelets per panicle 152.1±8.9 (10) 126.6±7.3 (13)* Number of fertile spikelets per panicle 143.9±7.9 (10) 33.8±2.4 (13)*** Number of primary branches per panicle 12.6±0.3 (10) 11.9±0.3 (13) Number of secondary branches per primary branch 2.0±0.1 (125) 1.6±0.1 (155)*** Length of the panicle axis (cm) 26.6±0.6 (8) 27.2±0.3 (13) -
Outer cortical cell and metaxylem element size, and number of cortical cell layers and of xylary poles were determined in digital microscope images of transverse sections taken 12 mm from the root tip. Number of adventitious and of lateral roots were determined in 2-week-old seedlings. Root elongation in 24 hours was monitored daily during a 1-month period. Leaf morphometric analyses were done on mature leaves of 6-month-old plants, unless otherwise indicated. Blade width and thickness, mesophyll cell size and number, metaxylem element size and vascular pattern parameters were measured through the middle region of the leaf blade. Blade thickness and mesophyll cell size and number (between the adaxial and the abaxial epidermis) were determined in the interveinal region using digital microscope images of transverse or longitudinal sections. Vascular pattern parameters were measured in dark-field microscopic digital pictures of cleared leaf preparations. The region of the leaf blade between the two most marginal adjacent large veins was excluded from all the measurements. Wild-type immature leaf populations were not significantly different in their length (0.75<P≤0.90) or width (0.90<P≤0.95) from the ral1 mature leaf population. Number of stem vascular bundles in the outer and inner ring were determined in digital microscope images of transverse sections through the apical part of the first internode of plants at the ripen-inflorescence stage. Morphometry of flowers was performed on ripe inflorescences after harvesting. Morphometric analysis using digital images was performed with the ImageJ 1.21 software. Results represent the mean±s.e.m. of populations of the size indicated in parenthesis. Asterisks indicate the significance of the difference between wild-type and ral1 populations as determined by single-factor ANOVA (root morphometry except for number of adventitious roots) or Student's t-test analysis (number of adventitious roots, leaf and flower morphometry): *0.01≤P<0.05, **0.001≤P<0.01, ***P<0.001. CVs, commissural veins, IVs, longitudinal veins; LVs, large veins; SVs, small veins.
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- Fig. 3.
Morphology and anatomy of wild-type and ral1 leaf blade commissural veins. (A,M,N) Wild type. (B-L,O,P) ral1. (A-L) Dark-field images of cleared leaves. (A) Uninterrupted commissural vein connecting two longitudinal veins. (B,C) Interrupted simple (B) or compound (C) commissural vein associated with one longitudinal vein. (D,E) Interrupted simple (D) or compound (E) commissural vein associated with two longitudinal veins. (F) Uninterrupted `Y' vein forming two connections with one longitudinal vein and one with the other. (G-K) Y veins showing interruptions at different positions. (L) Isolated patch of xylem elements (vascular island). (M-P) Paradermal sections through mature leaf blades. (M) Uninterrupted commissural vein connecting two longitudinal veins. (N) Detail of M. (O) Interrupted simple commissural vein associated with two longitudinal veins. (P) Detail of O. Scale bars: (A-L) 50 μm (M-P) 25 μm.
- Fig. 4.
Vascular development in wild type and ral1. (A-C,G,I,K,O-R,W,Y) Wild type. (D-F,H,J,L-N,S-V,X,Z) ral1. (A-F) Transverse sections through 2-week-old seedlings, 10 μm above the insertion of the first (A,D), second (B,E) and third (C,F) leaf primordium on the shoot apex (sixth, fifth and fourth leaf, respectively). (G-J) DR5-GUS (G,H) or Oshox1-GUS (I,J) expression in transverse vibratome sections (100 μm) through the shoot apex of 2-week-old seedlings. (K-N) Transverse section 200 μm above the shoot apex of 2-week-old seedlings showing Oshox1-GUS expression that identifies commissural veins developing in the fourth leaf primordium (third leaf). (O-V) DR5-GUS (O,P,S,T) or Oshox1-GUS (Q,R,U,V) expression in vascular bundles at a comparable stage of differentiation in the fourth (second protoxylem element stage; O,S,Q,U) and fifth (late metaxylem element stage; P,T,R,V) leaf primordium (third and second leaf, respectively). Xylem is oriented to the right. (W,X) Oshox1-GUS expression in mature embryos (dorsal view). (Y,Z) Schematic representation of the dorsal view of mature embryos showing the vascular system of the scutellum expressing (in blue) or not (in black) Oshox1-GUS. 1, 2, 3, first, second, and third leaf primordium, respectively; pp, protophloem; pv, provascular strand; px, protoxylem. Scale bars: (A-F) 100 μ m (G-N) 50 μm (O-V) 25 μm (W,X) 350 μm.
- Fig. 5.
Hormonal responses of wild type and ral1. (A,C,E-H,M-P,Q,S,U) Wild type. (B,D,I-L,R,T,V) ral1. (A-D) Seedlings 1 week (A,B) or 3 weeks (C,D) after callus induction on 2 mg/l 2,4-D. (E,F,I,J,M,N) Calli at the stage of transfer to the induction medium (E,I,M) and 3 weeks after (F,J,N) the transfer. Medium contained either 2 mg/l 2,4-D (E,F,I,J) or 1 mg/l 2,4-D (M,N). (G,H,K,L,O,P) Sections through the calli in F (G,H), in J (K,L), or in N (O,P). (Q-V) DR5-GUS expression in the root of 1-week-old seedlings grown for 24 hours on filter paper moistened with water (Q,R), with 0.1 (S,T) or 1 (U,V) μM NAA. (W,X) Frequency of shoot (W) or root (X) regeneration via somatic organogenesis in callus tissues grown on hormone-free medium (black diamond, wild type; black triangle, ral1) or on medium supplemented with cytokinin (black square, wild type; cross, ral1). The results represent the mean±s.e.m. of two separate experiments each performed on a population of 80-100 calli per genotype and per treatment. Difference between wild-type and ral1 populations as determined by repeated-measures analysis of variance (single-factor ANOVA) was significant (P<0.001) at all time points. (Y) Relative elongation over 24 hours of wild-type seminal (white boxes) and adventitious (grey boxes) roots and ral1 (black boxes) roots in the presence of 0.05 μM 2,4-D or 0.1 μM NAA. The results represent the mean±s.e.m. of two separate experiments each performed on a population of 20-35 seedlings per genotype and per treatment. Asterisks indicate the significance of difference between wild-type and ral1 populations as determined by repeated-measures analysis of variance (single-factor ANOVA). *0.01≤P<0.05, **0.001≤P<0.01. b, shoot base; pe, proembryonic structure; s, scutellum. Scale bars: (A-D,E,F,I,J,M,N) 2 mm (G,K) 100 μm (H,L,O,P) 25 μm (Q-V) 50 μm.
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