Photosynthesis in C3-C4 intermediate Moricandia species

Abstract

Evolution of C₄ photosynthesis is not distributed evenly in the plant kingdom. Particularly interesting is the situation in the Brassicaceae, because the family contains no C₄ species, but several C₃-C₄ intermediates, mainly in the genus Moricandia Investigation of leaf anatomy, gas exchange parameters, the metabolome, and the transcriptome of two C₃-C₄ intermediate Moricandia species, M. arvensis and M. suffruticosa, and their close C₃ relative M. moricandioides enabled us to unravel the specific C₃-C₄ characteristics in these Moricandia lines. Reduced CO₂ compensation points in these lines were accompanied by anatomical adjustments, such as centripetal concentration of organelles in the bundle sheath, and metabolic adjustments, such as the balancing of C and N metabolism between mesophyll and bundle sheath cells by multiple pathways. Evolution from C₃ to C₃-C₄ intermediacy was probably facilitated first by loss of one copy of the glycine decarboxylase P-protein, followed by dominant activity of a bundle sheath-specific element in its promoter. In contrast to recent models, installation of the C₃-C₄ pathway was not accompanied by enhanced activity of the C₄ cycle. Our results indicate that metabolic limitations connected to N metabolism or anatomical limitations connected to vein density could have constrained evolution of C₄ in Moricandia.

Keywords: Moricandia.; Bundle sheath; C3–C4 intermediacy; C4 photosynthesis; evolution; glycine decarboxylase.

Fig. 1.

C₃-C₄ intermediate species in the Brassicaceae. (A) Time-calibrated phylogeny of the Brassicaceae species selected for this study. The Moricandia species build one branch of the tree with an early separation of the C₃ species M. moricandioides and M. foetida, and the C₃–C₄ intermediate species M. suffruticosa, M spinosa, M. sinaica, M. nitens, and different M. arvensis lines. Additional independently established C₃–C₄ intermediate species are Diplotaxis tenuifolia and Brassica gravinae. The closest C₄ relative G. gynandra belongs to the Cleomaceae. C₃ species are in black; C₃–C₄ intermediate species in green, with gray for the potential intermediate D. erucoides; C₄ species in red. The scale bar is 5 Ma. (B) CO₂ compensation points determined from A-C_i curves in C₃ and C₃–C₄Moricandia lines compared with C₃ and C₄ species from the Cleomaceae. Significant differences were determined by ANOVA followed by Tukey’s HSD multiple comparison test with alpha ≤0.01.

Fig. 2.

Physiological features in the C₃ species M. moricandioides, and the C₃–C₄ intermediate species M. suffruticosa and M. arvensis line MOR1. (A) Representative examples of A-C_i curves from M. moricandioides, M. suffruticosa, and M. arvensis line MOR1 in comparison with the curve from the C₄ species G. gynandra. (B) CO₂ compensation points; (C) initial A-C_i slope; (D) assimilation rate under ambient conditions with c_a of 400 ppm; (E) δ¹³C signature of leaf material; (F) protein content per plant dry weight; (G) PEPC activity; (H) vein density in the top part of mature leaves. All box-whisker plots show data summarised from 8–10 biological replicates. The asterisks indicate significant differences of the C₃–C₄ intermediate species in comparison with the C₃ species M. moricandioides, as determined by a t-test (P≤0.01). Mmori, M. moricandioides; Marv, M. arvensis line MOR1; Msuff, M. suffruticosa. (This figure is available in colour at JXB online.)

Fig. 3.

Anatomy of the C₃ species M. moricandioides (A, D, G, J), and the C₃–C₄ intermediate species M.suffruticosa (B, E, H, K) and M.arvensis line MOR1 (C, F, I, L). (A–C) Top view of cleared leaves showing the general vein pattern; (D–F) overview of cross-sections; (G–I) close-up of the arrangement of chloroplasts in the bundle sheath cells; (J–L) transmission electron microscopy of bundle sheath cells with organelles arranged next to the vein cell. Arrow heads indicate vascular bundles. BS, bundle sheath cell; Xy, xylem cell; CC, companion cell.

Fig. 4.

Selected metabolites in the C₃ species M. moricandioides, and the C₃–C₄ intermediate species M. suffruticosa and M. arvensis line MOR1. The box-whisker plots represent summaries of 10–12 biological replicates. The asterisks indicate significant differences between the C₃–C₄ and the C₃ species as determined by a t-test (P≤0.01). Mmori, M. moricandioides; Marv, M. arvensis line MOR1; Msuff, M. suffruticosa. (This figure is available in colour at JXB online.)

Fig. 5.

Transcriptional changes in selected pathways. The heat maps indicate the log₂-fold changes in transcript level between C₃–C₄ species M. arvensis line MOR1 (left column) and M. suffruticosa (right column) and the C₃ species M. moricandioides. Blue indicates reduced transcript abundance in C₃–C₄, red indicates enhanced transcript abundance in C₃–C₄.

Fig. 6.

Comparison of the changes in transcript levels of the Moricandia species and Flaveria species with different degrees of C₄ features. The graphs show the relative amount (z-score normalised data of mean rpm values) from transcripts belonging to the selected pathways. The general trend in the transcription pattern of a pathway is indicated in red as the median of the individual transcripts values. Species abbreviations are: Mmori, M. moricandioides; Marv, M. arvensis; Msuff, M. suffruticosa; Fpri, F. pringlei; Frob, F. robusta; Fchl, F. chloraefolia; Fpub, F. pubescence; Fano, F. anomala; Fram, F. ramosissima; Fbro, F. brownii; Fbid, F. bidentis; Ftri, F. trinervia.

Fig. 7.

Phylogenetic tree of the glycine decarboxylase P-protein (GLDP) coding sequences in selected Brassicaceae. The GLDP copies from Arabidopsis thaliana are highlighted in blue, Moricandia species are in red, and Diplotaxis species are in green. Species abbreviations are: At, Arabidopsis thaliana; Alyt, Arabidopsis lyrata; Aalp, Arabis alpina; Bna, Brassica napus; Bo, B. oleraceae; Bra, B. rapa; Eutsa, Eutrema salsugineum; Thal, Thellungiella halophile; Csat, Camelina sativa; Carub, Capsella rubella; Thass, Tarenaya hassleriana; Mmori, Moricandia moricandioides; Mnit, Moricandia nitens; Marv, Moricandia arvansis line MOR1; Msuff, Moricandia suffruticosa; Dvim, Diplotaxis viminea; Dten, Diplotaxis tenuifolia; Dmur, Diplotaxis muralis). Branch support is determined by an approximate likelihood ratio test.

Fig. 8.

Model of metabolite shuttle network active between the mesophyll cells (left) and bundle sheath cells (right) of C₃–C₄ intermediate Moricandia species. The inactivity of the photorespiratory glycine decarboxylating complex in the mesophyll cells leads to glycine accumulation and transport to the bundle sheath. In the mitochondria of bundle sheath cells two molecules of glycine are converted by the GLD/SHMT complex to serine, CO₂, NH₃, and NADH. In the adjacent chloroplasts the bundle sheath Rubisco is exposed to enhanced CO₂ conditions. The imbalance created by parallel release of NH₃ requires further adjustment of C and N metabolism that is probably realised by a whole network of reactions, including additional shuttling of amino acids from the bundle sheath to the mesophyll (dark blue arrows), and re-shuttling of organic acids (light blue arrows). Enzymes highlighted in bold could be associated with an increased abundance of at least one transcript copy.