Use of diffusion magnetic resonance imaging to correlate the developmental changes in grape berry tissue structure with water diffusion patterns

Abstract

Background: Over the course of grape berry development, the tissues of the berry undergo numerous morphological transformations in response to processes such as water and solute accumulation and cell division, growth and senescence. These transformations are expected to produce changes to the diffusion of water through these tissues detectable using diffusion magnetic resonance imaging (MRI). To assess this non-invasive technique diffusion was examined over the course of grape berry development, and in plant tissues with contrasting oil content.

Results: In this study, the fruit of Vitis vinfera L. cv. Semillon at seven different stages of berry development, from four weeks post-anthesis to over-ripe, were imaged using diffusion tensor and transverse relaxation MRI acquisition protocols. Variations in diffusive motion between these stages of development were then linked to known events in the morphological development of the grape berry. Within the inner mesocarp of the berry, preferential directions of diffusion became increasingly apparent as immature berries increased in size and then declined as berries progressed through the ripening and senescence phases. Transverse relaxation images showed radial striation patterns throughout the sub-tissue, initiating at the septum and vascular systems located at the centre of the berry, and terminating at the boundary between the inner and outer mesocarp. This study confirms that these radial patterns are due to bands of cells of alternating width that extend across the inner mesocarp. Preferential directions of diffusion were also noted in young grape seed nucelli prior to their dehydration. These observations point towards a strong association between patterns of diffusion within grape berries and the underlying tissue structures across berry development. A diffusion tensor image of a post-harvest olive demonstrated that the technique is applicable to tissues with high oil content.

Conclusion: This study demonstrates that diffusion MRI is a powerful and information rich technique for probing the internal microstructure of plant tissues. It was shown that macroscopic diffusion anisotropy patterns correlate with the microstructure of the major pericarp tissues of cv. Semillon grape berries, and that changes in grape berry tissue structure during berry development can be observed.

Keywords: Development; Diffusion anisotropy; Diffusion tensor imaging; Grape berry; Nucellus; Nuclear magnetic resonance imaging; Olive; Seeds; Striation patterns; Vitis vinifera.

Figure 1

Anisotropic water diffusion due to diffusive restriction (two dimensional representation). Here water molecules (●) encounter cellular boundaries as they diffuse. As there are more cellular boundaries on the longitudinal axis than the lateral axis, water displacement along the longitudinal axis is reduced relative to the lateral axis (represented by the yellow ellipse).

Figure 2

Tissue regions of the grape berry (transverse plane). Here the five tissue regions of the grape berry are provided with reference to a transverse relaxation image (A) and a diffusion tensor image (B). Ex: exocarp, OM: outer mesocarp, IM: inner mesocarp, S: septum, SI: seed interior. The outer, black dashed curve indicates the border between the outer mesocarp and the inner mesocarp while the inner, black dashed curve indicates the border between the inner mesocarp and the septum.

Figure 3

The physical characteristics of the grape berries. The concentrations of soluble solids of the grape berries (Green ♦), as well as the fresh weight (Blue ▲) and dry weights (Red ▼) of the berries, are presented with respect to the number of days after flowering. A sigmoidal function (solid green curve) of the form a ₁ + (a ₂ - a ₁)/(1 + exp(-(DAF - x ₀)/w)) was fitted to the soluble solids values by nonlinear regression (adjusted R² = 0.99), where a ₁ = 26.1 (the approximate maximum soluble solids value), a ₂ = 3.9 (the approximate minimum soluble solids value), x ₀ = 69.7 (the inflection point) and w =7.4 (the change in DAF which yielded the greatest change in the soluble solids value). The error bars are given by the standard deviation of soluble solids values at each time point.

Figure 4

Transverse relaxation images of grape berries at seven different stages of berry development (transverse plane). The images include three pre-véraison grapes, at 28 DAF (A, voxel size 59 × 59 × 1000 μm), 41 DAF (B, voxel size 78 × 78 × 1000 μm) and 55 DAF (C, voxel size 78 × 78 × 1000 μm), a grape undergoing véraison at 70 DAF (D, voxel size 82 × 82 × 1000 μm), a ripening grape at 85 DAF (E, voxel size 74 × 74 × 1000 μm), a grape which is at oenological maturity at 95 DAF (F, voxel size 63 × 63× 1000 μm) and a post-maturity berry at 109 DAF (G, voxel size 86 × 86 × 1000 μm). The transverse relaxation values are indicated by the colour bar to the right of the figure. Scale bar: 3 mm.

Figure 5

Transverse relaxation images of grape berries at seven different stages of berry development (longitudinal plane) . The images include three pre-véraison grapes, at 28 DAF (A, voxel size 59 × 59 × 1000 μm), 41 DAF (B, voxel size 78 × 78 × 1000 μm) and 55 DAF (C, voxel size 78 × 78 × 1000 μm), a grape undergoing véraison at 70 DAF (D, voxel size 133 × 133 × 1000 μm), a ripening grape at 85 DAF (E, voxel size 82 × 82 × 1000 μm), a grape which is at oenological maturity at 95 DAF (F, voxel size 63 × 63 × 1000 μm) and a post-maturity berry at 109 DAF (G, voxel size 86 × 86 × 1000 μm). The transverse relaxation values are indicated by the colour bar to the right of the figure. Scale bar: 3 mm.

Figure 6

Transverse (T ₂ ) relaxation of the major tissue groups of the grape berry with respect to time (transverse plane). The transverse relaxation values for the exocarp (■), outer mesocarp (Red ●), inner mesocarp (Blue ▲) and septum (Pink ▼). The error bars are given by the standard deviation of the transverse relaxation values at each time point.

Figure 7

DT images of grape berries at seven different stages of berry development (transverse plane). The images include three pre-véraison grapes, at 28 DAF (A, voxel size 117 × 117 × 1000 μm), 41 DAF (B, voxel size 156 × 156 × 1000 μm) and 55 DAF (C, voxel size 156 × 156 × 1000 μm), a grape undergoing véraison at 70 DAF (D, voxel size 164 × 164 × 1000 μm), a ripening grape at 85 DAF (E, voxel size 148 × 148 × 1000 μm), a grape at oenological maturity at 95 DAF (F, voxel size 125 × 125 × 1000 μm) and a post-maturity berry, at 109 DAF (G, voxel size 171 × 171 × 1000 μm). The colours in the figure indicate the direction of least restricted diffusion, as indicated by the image at the bottom right of the figure. Scale bar: 3 mm.

Figure 8

DT images of grape berries at five different stages of berry development (longitudinal plane). The images include a pre-véraison grape at 55 DAF (A, voxel size 156 × 156 × 1000 μm), a grape undergoing véraison at 70 DAF (B, voxel size 164 × 164 × 1000 μm), a ripening grape at 85 DAF (C, voxel size 172 × 172 × 1000 μm), a grape which is at oenological maturity at 95 DAF (D, voxel size 125 × 125 × 1000 μm) and a post-maturity berry at 109 DAF (E, voxel size 172 × 172 × 1000 μm). No images are available for 28 and 41 DAF. The colours in the figure indicate the direction of least restricted diffusion, as indicated by the image in the bottom right side of the figure. Images are not available for 28 and 41 DAF. Scale bar: 3 mm.

Figure 9

Diffusion vector field map overlaying the S ₀ image of a grape berry 41 DAF (transverse plane). The diffusion vectors (blue arrows) indicate the direction of least restricted diffusion in each voxel. Voxel size: 156 × 156 × 1000 μm, bar length: 1000 μm.

Figure 10

Mean ADC of the major tissue groups of the grape berry with respect to time and total soluble solids . The mean ADC for the exocarp (■), outer mesocarp (Red ●), inner mesocarp (Blue ▲) and septum (Pink ▼) decrease sigmoidally (adjusted R² = 0.99) with respect to sigmoidally increasing (adjusted R² = 0.99) dissolved solids content (Green ♦). The error bars are given by the standard deviation of the mean ADC at each time point.

Figure 11

Mean ADC map of a grape berry 55 DAF (transverse plane) . Voxel size 78 × 78 × 1000 μm, bar length: 3000 μm.

Figure 12

Diffusion vector field map overlaying the S ₀ image of a grape berry 109 DAF (transverse plane). The orientation of diffusion vectors are indicated by the blue arrows. There was a loss of diffusion-weighted signal in the region denoted by the red dashed line. Voxel size: 133 × 133 × 1000 μm, bar length: 1000 μm.

Figure 13

Diffusion vector field map overlaying the S ₀ image of a grape berry seed interior 28 DAF (transverse plane) . The diffusion vectors (blue arrows) indicate the direction of least restricted diffusion. Voxel size: 117 × 117 × 1000 μm, bar length: 1000 μm.

Figure 14

DT and transverse relaxation images of a postharvest olive (transverse plane). The DT image (A, voxel size 390 × 390 × 1000 μm) and diffusion vector map (B, voxel size 390 × 390 × 1000 μm) of an olive pericarp. Bar length 1000 μm.

Figure 15

Confocal micrograph of the pericarp of a grape berry prior to véraison 41 DAF (transverse plane). The image was acquired using a confocal microscope (LSM5 Pascal; Zeiss, Germany) which employed a 488 nm Argon laser and a 10 × objective Plan-Apochromatic lens. Bar length 1000 μm.

Figure 16

Confocal micrograph of the pericarp of a grape berry undergoing véraison 55 DAF (transverse plane). The image was acquired using a confocal microscope (LSM5 Pascal; Zeiss, Germany) which employed a 488 nm Argon laser and a 10 × objective Plan-Apochromatic lens. Bar length 1000 μm.