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Mapping by VESGEN of Leaf Venation Patterning in Arabidopsis thaliana with Bioinformatic Dimensions of Gene Expression

Patricia Parsons-Wingerter
Patricia Parsons-Wingerter
, 
Mary B. Vickerman
Mary B. Vickerman
, 
Anna-Lisa Paul
Anna-Lisa Paul
 y 
Robert J. Ferl
Robert J. Ferl
   | 18 ene 2022
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Article Category: Research Article
Publicado en línea: 18 ene 2022
Páginas: 68 - 81
DOI: https://doi.org/10.2478/gsr-2014-0006
© 2014 Patricia Parsons-Wingerter et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Figure 1.

Botanical rules for venation patterning by branching order in higher leaves. Large structural (dendritic) veins of primary (1°) and secondary (2°) branching orders in a dicot angiosperm leaf, such as Acer argutum, are organized as a dendritic tree in which 1° veins extend from the leaf’s base to tip (apex), and 2° costal (ribbed) veins branch laterally as parallel offshoots from the 1° vein(s). Cleared leaf images are reproduced from Ellis et al. (2009). Structural veins tend to taper progressively. The reticulate network of orders tertiary and greater (≥ 3°) is composed of smaller, intercostal, connected veins, including end veinlets that terminate at pores where photosynthesis occurs. The vascular dendritic tree connects continuously with the reticulate network to form a unique leaf venation pattern that for angiosperms, is an accepted species taxonomic identifier. Acer argutum is classified as a palmate dicot leaf because of its multiple 1° veins. (b) For pinnate dicot leaves represented by the three leaf schematics (reproduced from (Roth-Nebelsick et al., 2001)) that include ISS model organism Arabidopsis thaliana (Figures 2-4), 2° veins branch from a single 1° vein. Arabidopsis juvenile leaves display brochidodromous leaf venation, whereas adult leaves are semi-craspedodromous (Kang and Dengler, 2004). Overall, venation patterns in angiosperm leaves are organized as tree-network composites.
Botanical rules for venation patterning by branching order in higher leaves. Large structural (dendritic) veins of primary (1°) and secondary (2°) branching orders in a dicot angiosperm leaf, such as Acer argutum, are organized as a dendritic tree in which 1° veins extend from the leaf’s base to tip (apex), and 2° costal (ribbed) veins branch laterally as parallel offshoots from the 1° vein(s). Cleared leaf images are reproduced from Ellis et al. (2009). Structural veins tend to taper progressively. The reticulate network of orders tertiary and greater (≥ 3°) is composed of smaller, intercostal, connected veins, including end veinlets that terminate at pores where photosynthesis occurs. The vascular dendritic tree connects continuously with the reticulate network to form a unique leaf venation pattern that for angiosperms, is an accepted species taxonomic identifier. Acer argutum is classified as a palmate dicot leaf because of its multiple 1° veins. (b) For pinnate dicot leaves represented by the three leaf schematics (reproduced from (Roth-Nebelsick et al., 2001)) that include ISS model organism Arabidopsis thaliana (Figures 2-4), 2° veins branch from a single 1° vein. Arabidopsis juvenile leaves display brochidodromous leaf venation, whereas adult leaves are semi-craspedodromous (Kang and Dengler, 2004). Overall, venation patterns in angiosperm leaves are organized as tree-network composites.

Figure 2.

Arabidopsis as model organism of leaf venation patterning in dicot angiosperms. Illustrations of the adult plant by (a) watercolor with roots (Sturm, 1796) and (b) photograph (Page and Grossniklaus, 2002). (c) Rosette stage of young juvenile and adult leaves (Paul et al., 2001). (d) Graph reproduced from Kang and Dengler (2004) quantifies the developmental time course of expansion for the Arabidopsis leaf lamina and petiole of juvenile Leaf 1 and adult Leaf 8. (e) Arabidopsis seedlings cultivated within NASA’s Advanced Biological Research System (ABRS, see Paul et al., 2012; Paul et al., 2013b).
Arabidopsis as model organism of leaf venation patterning in dicot angiosperms. Illustrations of the adult plant by (a) watercolor with roots (Sturm, 1796) and (b) photograph (Page and Grossniklaus, 2002). (c) Rosette stage of young juvenile and adult leaves (Paul et al., 2001). (d) Graph reproduced from Kang and Dengler (2004) quantifies the developmental time course of expansion for the Arabidopsis leaf lamina and petiole of juvenile Leaf 1 and adult Leaf 8. (e) Arabidopsis seedlings cultivated within NASA’s Advanced Biological Research System (ABRS, see Paul et al., 2012; Paul et al., 2013b).

Figure 3.

Rules-based mapping of environmental effects on venation patterning that are fundamental to photosynthetic function. (a, b) Arabidopsis juvenile Leaf 2 cultured in the ISS microgravity environment from a spaceflight experiment flown on NASA STS-130 is compared to wild-type ground control. When analyzed by VESGEN according to vertebrate vascular branching rules (second column), the pronounced tapering of vessel diameter in the 1° midvein resulted in division of the vessel into four segments (Legend: colors corresponding to branching generations G1-G7). Altering the branching rules according to dicot leaf venation patterning (third column) resulted in successful VESGEN segmentation into venous structural orders (1°, red; 2°, yellow) and reticulate orders (3°, turquoise; 4°, purple). As a second mapping option, vessels were grouped (fourth column) by VESGEN into structural veins (1°-2°) and reticulate veins (3°-4°). By VESGEN analysis, overall differences between the two juvenile leaves were large for the functionally important smaller (reticulate) veins. (a, b) scalebar = 1 mm.
Rules-based mapping of environmental effects on venation patterning that are fundamental to photosynthetic function. (a, b) Arabidopsis juvenile Leaf 2 cultured in the ISS microgravity environment from a spaceflight experiment flown on NASA STS-130 is compared to wild-type ground control. When analyzed by VESGEN according to vertebrate vascular branching rules (second column), the pronounced tapering of vessel diameter in the 1° midvein resulted in division of the vessel into four segments (Legend: colors corresponding to branching generations G1-G7). Altering the branching rules according to dicot leaf venation patterning (third column) resulted in successful VESGEN segmentation into venous structural orders (1°, red; 2°, yellow) and reticulate orders (3°, turquoise; 4°, purple). As a second mapping option, vessels were grouped (fourth column) by VESGEN into structural veins (1°-2°) and reticulate veins (3°-4°). By VESGEN analysis, overall differences between the two juvenile leaves were large for the functionally important smaller (reticulate) veins. (a, b) scalebar = 1 mm.

Figure 4.

Integrated mapping in arabidopsis of leaf venation pattern with bioinformatic dimension of gene expression by rules-based VESGEN Analysis. Patterns of differentiated xylem (a, f) with AtHB-8::GUS expression (b, g) in the normally developing arabidopsis adult leaf (Leaf 8) at Day 2 (D2) and Day 8 (D8) of normal development reproduced from a study by Kang and Dengler (2004) were mapped by VESGEN software (c-e, h-j). (c, h) Large structural veins (orders 1° and 2°) and small reticular veins (3° and ≥ 4°) are mapped with botanical rules summarized in Figure 1. Note the large increase in leaf size and vascular expansion from Day 2 to Day 8 (h). (d, i) Euclidean distance mapping by VESGEN quantifies the local thickness of vessel diameter throughout the vascular tree-network composite. Black indicates enclosed avascular spaces. To demonstrate our proposed VESGEN bioinformatic capability, the time-dependent localized expression of HD-Zip class III transcription factor arabidopsis homeobox gene-8 (AtHB-8::GUS, b, g) was mapped into vascular patterns automatically grouped by VESGEN into structural (1°-2°) and reticulate (3°-4°) vein orders (e, j). The mapping for Day 2 (e) displays the local intensity of AtHB-8::GUS expression throughout the leaf lamina as a function of the venation architecture and vessel branching order illustrated in panel c. Red intensity levels quantify AtHB-8::GUS expression localized within structural veins of orders 1°-2°; yellow intensity levels, within reticulate veins of orders 3°-4°; and gray intensity levels, to AtHB-8::GUS expression within the extravascular leaf lamina (intensities brightened for visibility). (j) AtHB-8::GUS expression at Day 8 is now restricted to the still-expanding basal region. Quantification corresponding to these mappings for D2 and D8 is summarized in Table 2. CAPTION NOTES from Kang and Dengler (2004): (b) 1, first-formed secondary vein loop; 2, second-formed secondary vein; black arrowhead, intercalated secondary vein; black arrow, smaller-diameter “connector” joining adjacent secondary vein; (g) inset showing absence of AtHB-8::GUS expression from leaf apex. a, bar = 200 μm; f, bar = 1 mm
Integrated mapping in arabidopsis of leaf venation pattern with bioinformatic dimension of gene expression by rules-based VESGEN Analysis. Patterns of differentiated xylem (a, f) with AtHB-8::GUS expression (b, g) in the normally developing arabidopsis adult leaf (Leaf 8) at Day 2 (D2) and Day 8 (D8) of normal development reproduced from a study by Kang and Dengler (2004) were mapped by VESGEN software (c-e, h-j). (c, h) Large structural veins (orders 1° and 2°) and small reticular veins (3° and ≥ 4°) are mapped with botanical rules summarized in Figure 1. Note the large increase in leaf size and vascular expansion from Day 2 to Day 8 (h). (d, i) Euclidean distance mapping by VESGEN quantifies the local thickness of vessel diameter throughout the vascular tree-network composite. Black indicates enclosed avascular spaces. To demonstrate our proposed VESGEN bioinformatic capability, the time-dependent localized expression of HD-Zip class III transcription factor arabidopsis homeobox gene-8 (AtHB-8::GUS, b, g) was mapped into vascular patterns automatically grouped by VESGEN into structural (1°-2°) and reticulate (3°-4°) vein orders (e, j). The mapping for Day 2 (e) displays the local intensity of AtHB-8::GUS expression throughout the leaf lamina as a function of the venation architecture and vessel branching order illustrated in panel c. Red intensity levels quantify AtHB-8::GUS expression localized within structural veins of orders 1°-2°; yellow intensity levels, within reticulate veins of orders 3°-4°; and gray intensity levels, to AtHB-8::GUS expression within the extravascular leaf lamina (intensities brightened for visibility). (j) AtHB-8::GUS expression at Day 8 is now restricted to the still-expanding basal region. Quantification corresponding to these mappings for D2 and D8 is summarized in Table 2. CAPTION NOTES from Kang and Dengler (2004): (b) 1, first-formed secondary vein loop; 2, second-formed secondary vein; black arrowhead, intercalated secondary vein; black arrow, smaller-diameter “connector” joining adjacent secondary vein; (g) inset showing absence of AtHB-8::GUS expression from leaf apex. a, bar = 200 μm; f, bar = 1 mm

Quantification of venation pattern in Arabidopsis Adult Leaf 8 by VESGEN analysis. (Large) structural vein orders, 1°-2°; (small) reticulate vein orders, ≥ 3°. Symbols and units: fractal dimension (Df), unitless (of skeletonized images); vessel area density (Av),µm2/µm2; vessel number density (Nv),µm-2 [total number of vessels in leaf lamina (Nr)]; vessel diameter (Dv),µm.

AnalysisDayVesselGroupDfAvNv[Nr]Dv
Figure 42all orders1.380.3532.68E-4[119]–
Kang & Dengler 20041°-2°–0.2891.53E-4[68]1°, 50; 2°, 17
≥ 3°–0.0631.15E-4[51]3°, 11; 4°, 8
8all orders1.470.2986.2E-6[411]–
1°-2°–0.1432.3E-6[153]1°, 131; 2°, 84
≥ 3°–0.1573.9E-6[255]3°, 72; 4°, 58

Quantification of venation patterning in Arabidopsis Juvenile Leaf 2 by VESGEN analysis. (Large) structural vein orders, 1°-2°; (small) reticulate vein orders, ≥ 3°. Symbols and units: fractal dimension (Df), unitless (of skeletonized images); vessel area density (Av),µm2/µm2; vessel number density (Nv),µm-2 [total number of vessels in leaf lamina (Nr)]; vessel diameter (Dv),µm.

AnalysisDayVesselDfAvNvDv
Group[Nr]
Figure 37all orders1.340.08132.99E-5[149]–
Spaceflight1°-2°–0.05691.24E-51°, 19; 2°, 10
STS-130[62]
≥ 3°–0.02441.74E-5[87]3°, 8; 4°, 7
Ground control7all orders1.320.06732.07E-5[110]–
1°-2°–0.05591.29E-5[69]1°, 27; 2°, 11
≥ 3°–0.01207.70E-6[41]3°, 8; 4°, 5
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