Light sheet microscopy reveals more gradual light attenuation in light-green versus dark-green soybean leaves

Abstract

Light wavelengths preferentially absorbed by chlorophyll (chl) often display steep absorption gradients. This over-saturates photosynthesis in upper chloroplasts and deprives lower chloroplasts of blue and red light. Reducing chl content could create a more even leaf light distribution and thereby increase leaf light-use efficiency and overall canopy photosynthesis. This was tested on soybean cultivar 'Clark' (WT) and a near-isogenic chl b deficient mutant, Y11y11, grown in controlled environment chambers and in the field. Light attenuation was quantified using a novel approach involving light sheet microscopy. Leaf adaxial and abaxial surfaces were illuminated separately with blue, red, and green wavelengths, and chl fluorescence was detected orthogonally to the illumination plane. Relative fluorescence was significantly greater in deeper layers of the Y11y11 mesophyll than in WT, with the greatest differences in blue, then red, and finally green light when illuminated from the adaxial surface. Modeled relative photosynthesis based on chlorophyll profiles and Beer's Law predicted less steep gradients in mutant relative photosynthesis rates compared to WT. Although photosynthetic light-use efficiency was greater in the field-grown mutant with ~50% lower chl, light-use efficiency was lower in the mutant when grown in chambers where chl was ~80% reduced. This difference is probably due to pleiotropic effects of the mutation that accompany very severe reductions in chlorophyll and may warrant further testing in other low-chl lines.

Keywords: Chlorophyll; Glycine max; leaf light environment; light sheet microscopy; light use efficiency; photosynthesis; photosynthetic efficiency; soybean..

Fig. 1.

Fluorescence profiles of chamber-grown WT (ABCGHI) and Y11y11 (DEFJKL) cross-sections within a z-stack when illuminated from the adaxial (A–F) or abaxial (G–L) surface with blue (445nm; A, D, G, J), red (638nm; B, E, H, K), and green (561nm; C, F, I, L) lasers using light sheet microscopy. Fluorescence was falsely colored to represent the illumination wavelength. Scale bar in (A) = 50 µm. Pixel size = 0.23 µm.

Fig. 2.

Relative fluorescence with distance from the adaxial surface. WT (black) and Y11y11 (grey) chamber-grown leaves were illuminated with blue (445nm; A, B), red (638nm; C, D), and green (561nm; E, F) lasers from the adaxial (A, C, E) and abaxial (B, D, F) surface using light sheet microscopy. Mean fluorescence (n=6) is shown every 0.23 µm. Error bars are indicated every 10 µm. Asterisks represent significant differences at P<0.05.

Fig. 3.

Relative fluorescence with distance from the adaxial surface. WT (black) and Y11y11 (grey) field-grown leaves were illuminated with 405nm (A, B), 488nm (C, D), 638nm (E, F), and 561nm (G, H) lasers from the adaxial (A, C, E, G) and abaxial (B, D, F, H) surface using light sheet microscopy. Means (n=6) are shown every 0.23 µm. Error bars are indicated every 10 µm. Asterisks represent significant differences at P<0.05.

Fig. 4.

Epi-fluorescence, chl content, and relative Rubisco profiles in chamber-grown (A, C, E) and field-grown (B, D, F) WT (black) and Y11y11 (grey) leaves. Relative chl distribution from epi-fluorescence measurements (A, B) was converted to absolute chl content (C, D) based on total leaf chl content on an area basis. Relative Rubisco (E, F) was determined from previous relationships between Rubisco per chl versus cumulative chl (Evans, 1995, 1999). Sample size in (A, B) was n=6 except for chamber-grown Y11y11 (n=5). Error bars represent standard errors of the means.

Fig. 5.

Light availability, absorption, and relative photosynthesis profiles in chamber-grown (A, C, E) and field-grown (B, D, F) WT (black) and Y11y11 (grey) leaves. The amount of light available (A, B) and absorbed (C, D) in each layer were used to determine relative photosynthesis profiles (E, F).

Fig. 6.

Gas exchange from light response curves. A (A, B), g _s (C, D), and C _i (E, F) were measured as a function of absorbed photosynthetic photon flux density (PPFD_abs) in WT (black) and Y11y11 (grey) chamber-grown (A, C, E) and field-grown (B, D, F) soybean. Solid lines represent the mean and dashed lines represent the 95% confidence intervals (n=6 in A, C, E; n=4 in B, D, F).

Fig. 7.

Chl fluorescence parameters from light response curves. F _v′/F _m′ (A, B), ϕPSII (C, D), NPQ (E, F), and q _P (G, H) were measured as a function of absorbed photosynthetic photon flux density (PPFD_abs) in WT (black) and Y11y11 (grey) chamber-grown (A, C, E, G) and field-grown (B, D, F, H) soybean. Solid lines represent the mean and dashed lines represent the 95% confidence intervals (n=6).