Photosynthesis-related characteristics of the midrib and the interveinal lamina in leaves of the C3-CAM intermediate plant Mesembryanthemum crystallinum

Abstract

Background and aims: Leaf veins are usually encircled by specialized bundle sheath cells. In C4 plants, they play an important role in CO2 assimilation, and the photosynthetic activity is compartmentalized between the mesophyll and the bundle sheath. In C3 and CAM (Crassulacean acid metabolism) plants, the photosynthetic activity is generally attributed to the leaf mesophyll cells, and the vascular parenchymal cells are rarely considered for their role in photosynthesis. Recent studies demonstrate that enzymes required for C4 photosynthesis are also active in the veins of C3 plants, and their vascular system contains photosynthetically competent parenchyma cells. However, our understanding of photosynthesis in veins of C3 and CAM plants still remains insufficient. Here spatial analysis of photosynthesis-related properties were applied to the midrib and the interveinal lamina cells in leaves of Mesembryanthemum crystallinum, a C3-CAM intermediate plant.

Methods: The midrib anatomy as well as chloroplast structure and chlorophyll fluorescence, diurnal gas exchange profiles, the immunoblot patterns of PEPC (phosphoenolpyruvate carboxylase) and RubisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase), H2O2 localization and antioxidant enzyme activities were compared in the midrib and in the interveinal mesophyll cells in leaves of C3 and CAM plants.

Key results: Leaf midribs were structurally competent to perform photosynthesis in C3 and CAM plants. The midrib chloroplasts resembled those in the bundle sheath cells of C4 plants and were characterized by limited photosynthetic activity.

Conclusions: The metabolic roles of midrib chloroplasts differ in C3 and CAM plants. It is suggested that in leaves of C3 plants the midrib chloroplasts could be involved in the supply of CO2 for carboxylation, and in CAM plants they could provide malate to different metabolic processes and mediate H2O2 signalling.

Keywords: Antioxidant enzymes; Mesembryanthemum crystallinum; chloroplast structure; gas exchange; interveinal lamina tissues; midrib; photosynthetic/photochemical activity.

Fig. 1.

Morphology of Mesembryanthemum crystallinum leaf: (A) abaxial side of the leaf of the third leaf pair and the analysed leaf areas; (B) a view of abaxial epidermis from the interveinal area of the leaf blade.

Fig. 2.

The midrib anatomy (A), and amounts of callose (B) and lignin (C) in the leaf midribs of C₃ and CAM Mesembryanthemum crystallinum plants. Scale bar = 100 μm for all images. (A) A representative example of three experiments with similar outcomes is shown. Three C₃ and CAM plants were used per experiment (n = 9). (B, C) The amounts of callose and lignin were expressed in arbitrary units (a.u.) of fluorescence intensity (FI). Data are means ± s.d. from n = 9. * indicates significant difference at P < 0.05 between CAM and C₃ plants for a given tissue type of the leaf midrib.

Fig. 3.

Daily changes of stomatal conductance (A) and net photosynthesis of leaf lamina (B) and midrib (C) in leaves of C₃ and CAM Mesembryanthemum crystallinum plants. (A–C) Each curve is representative of three replicate runs with ±5 % (n = 3).

Fig. 4.

Images of a representative midrib (A) and photosynthetically active radiation (PAR) absorptivity of C₃ (B) and CAM (C) Mesembryanthemum crystallinum leaves. The fluorescence micrograph (A) shows cells of (a) parenchyma, (b) bottom collenchyma, (c) xylem and (d) upper collenchyma in a leaf midrib. This picture is representative for C₃ and CAM leaves. Arrows point to the red fluorescence of chloroplasts in the bottom (b) and upper (d) collenchyma cells as well as in mesophyll (a) and xylem (c) parenchyma cells. In C₃ (B) and CAM (C) plants, the interveinal leaf laminae and veins (arrows) differ in PAR absorptivity. The mean PAR absorptivity values in the laminae and veins are shown in the white and black boxed areas, respectively. Scale bars represent 100 μm. (A–C) Images show representative examples from three experiments with similar results. A minimum of three C₃ and CAM plants were used per experiment.

Fig. 5.

Chloroplasts in the cells of leaf lamina (A, C) and midrib (B, D) of C₃ (A, B) and CAM (C, D) Mesembryanthemum crystallinum plants. (A–D) Representative examples of three experiments with similar results.

Fig. 6.

Visualization of H₂O₂ by DAB staining in detached leaves of C₃ (A) and CAM (B) plants, and APX (C) and CAT (D) activities in leaf laminae and midribs. The inset in (B) shows details of the DAB staining effects. (A, B) Representative examples of three experiments with similar outcomes. A minimum of three C₃ and CAM plants were used per experiment. (C, D) The APX and CAT data are means ± s.d. from three experiments with two replicates (n = 6).

Fig. 7.

Western blots of RubisCO and PEPC in laminae and midribs of leaves of C₃ and CAM Mesembryanthemum crystallinum. MM, molecular mass markers. Images show representative examples from three experiments with similar results. A minimum of three C₃ and CAM plants were used per experiment.