Tribulus (Zygophyllaceae) as a case study for the evolution of C2 and C4 photosynthesis.

C2 photosynthesis is a photosynthetic pathway in which photorespiratory CO2 release and refixation are enhanced in leaf bundle sheath (BS) tissues. The evolution of C2 photosynthesis has been hypothesized to be a major step in the origin of C4 photosynthesis, highlighting the importance of studying C2 evolution. In this study, physiological, anatomical, ultrastructural, and immunohistochemical properties of leaf photosynthetic tissues were investigated in six non-C4 Tribulus species and four C4 Tribulus species. At 42°C, T. cristatus exhibited a photosynthetic CO2 compensation point in the absence of respiration (C*) of 21 µmol mol-1, below the C3 mean C* of 73 µmol mol-1. Tribulus astrocarpus had a C* value at 42°C of 55 µmol mol-1, intermediate between the C3 species and the C2 T. cristatus. Glycine decarboxylase (GDC) allocation to BS tissues was associated with lower C*. Tribulus cristatus and T. astrocarpus allocated 86% and 30% of their GDC to the BS tissues, respectively, well above the C3 mean of 11%. Tribulus astrocarpus thus exhibits a weaker C2 (termed sub-C2) phenotype. Increased allocation of mitochondria to the BS and decreased length-to-width ratios of BS cells, were present in non-C4 species, indicating a potential role in C2 and C4 evolution.

The genome of Eleocharis vivipara elucidates the genetics of C3-C4 photosynthetic plasticity and karyotype evolution in the Cyperaceae.

Eleocharis vivipara, an amphibious sedge in the Cyperaceae family, has several remarkable properties, most notably its alternate use of C3 photosynthesis underwater and C4 photosynthesis on land. However, the absence of genomic data has hindered its utility for evolutionary and genetic research. Here, we present a high-quality genome for E. vivipara, representing the first chromosome-level genome for the Eleocharis genus, with an approximate size of 965.22 Mb mainly distributed across 10 chromosomes. Its Hi-C pattern, chromosome clustering results, and one-to-one genome synteny across two subgroups indicates a tetraploid structure with chromosome count 2n = 4x = 20. Phylogenetic analysis suggests that E. vivipara diverged from Cyperus esculentus approximately 32.96 million years ago (Mya), and underwent a whole-genome duplication (WGD) about 3.5 Mya. Numerous fusion and fission events were identified between the chromosomes of E. vivipara and its close relatives. We demonstrate that E. vivipara has holocentromeres, a chromosomal feature which can maintain the stability of such chromosomal rearrangements. Experimental transplantation and cross-section studies showed its terrestrial culms developed C4 Kranz anatomy with increased number of chloroplasts in the bundle sheath (BS) cells. Gene expression and weighted gene co-expression network analysis (WGCNA) showed overall elevated expression of core genes associated with the C4 pathway, and significant enrichment of genes related to modified culm anatomy and photosynthesis efficiency. We found evidence of mixed nicotinamide adenine dinucleotide - malic enzyme and phosphoenolpyruvate carboxykinase type C4 photosynthesis in E. vivipara, and hypothesize that the evolution of C4 photosynthesis predates the WGD event. The mixed type is dominated by subgenome A and supplemented by subgenome B. Collectively, our findings not only shed light on the evolution of E. vivipara and karyotype within the Cyperaceae family, but also provide valuable insights into the transition between C3 and C4 photosynthesis, offering promising avenues for crop improvement and breeding.