Insights into Regulation of C2 and C4 Photosynthesis in Amaranthaceae/Chenopodiaceae Using RNA-Seq

Amaranthaceae (incl. Chenopodiaceae) shows an immense diversity of C4 syndromes. More than 15 independent origins of C4 photosynthesis, and the largest number of C4 species in eudicots signify the importance of this angiosperm lineage in C4 evolution. Here, we conduct RNA-Seq followed by comparative transcriptome analysis of three species from Camphorosmeae representing related clades with different photosynthetic types: Threlkeldia diffusa (C3), Sedobassia sedoides (C2), and Bassia prostrata (C4). Results show that B. prostrata belongs to the NADP-ME type and core genes encoding for C4 cycle are significantly upregulated when compared with Sed. sedoides and T. diffusa. Sedobassia sedoides and B. prostrata share a number of upregulated C4-related genes; however, two C4 transporters (DIT and TPT) are found significantly upregulated only in Sed. sedoides. Combined analysis of transcription factors (TFs) of the closely related lineages (Camphorosmeae and Salsoleae) revealed that no C3-specific TFs are higher in C2 species compared with C4 species; instead, the C2 species show their own set of upregulated TFs. Taken together, our study indicates that the hypothesis of the C2 photosynthesis as a proxy towards C4 photosynthesis is questionable in Sed. sedoides and more in favour of an independent evolutionary stable state.

Insights into regulation of C2 and C4 photosynthesis in Amaranthaceae/Chenopodiaceae using RNA-Seq

Amaranthaceae (incl. Chenopodiaceae) show an immense diversity of C4 syndromes. More than 15 independent origins of C4 photosynthesis, partly in halophytic and/or succulent lineages, and the largest number of C4 species in eudicots signify the importance of this angiosperm lineage in C4 evolution. Here, we conduct RNA-Seq followed by comparative transcriptome analysis of three species from Camphorosmeae representing related clades with different photosynthetic types: Threlkeldiadiffusa (C3), Sedobassiasedoides (C2), and Bassiaprostrata (C4). Results show that B.prostrata belongs to the NADP–ME type and core genes encoding for C4 cycle are significantly up–regulated when compared to Sed.sedoides and T.diffusa, Sedobassiasedoides and B.prostrata share a number of up–regulated C4–related genes, however, two C4 transporters (DIT and TPT) are found significantly up–regulated only in Sed. sedoides. Combined analysis of transcription factors (TFs) of the closely related lineages (Camphorosmeae and Salsoleae) revealed that no C3 specific TFs is higher in C2 species as compared to C4 species, instead the C2 species show their own set of up–regulated TFs. Taken together, our study indicates that the hypothesis of the C2 photosynthesis as a proxy towards C4 photosynthesis is questionable in Sed.sedoides and more in favour of an independent evolutionary stable–state.

Upregulation of C4 characteristics does not consistently improve photosynthetic performance in intraspecific hybrids of a grass

C4 photosynthesis is thought to have evolved via intermediate stages, with changes towards the C4 phenotype gradually enhancing photosynthetic performance. This hypothesis is widely supported by modelling studies, but experimental tests are missing. Mixing of C4 components to generate artificial intermediates can be achieved via crossing, and the grass Alloteropsis semialata represents an outstanding system since it includes C4 and non-C4 populations. Here, we analyse F1 hybrids between C3 and C4, and C3+C4 and C4 genotypes to determine whether the acquisition of C4 characteristics increases photosynthetic performance. The hybrids have leaf anatomical characters and C4 gene expression profiles that are largely intermediate between those of their parents. Carbon isotope ratios are similarly intermediate, which suggests that a partial C4 cycle coexists with C3 carbon fixation in the hybrids. This partial C4 phenotype is associated with C4-like photosynthetic efficiency in C3+C4 x C4, but not in C3 x C4 hybrids, which are overall less efficient than both parents. Our results support the hypothesis that the photosynthetic gains from the upregulation of C4 characteristics depend on coordinated changes in anatomy and biochemistry. The order of acquisition of C4 components is thus constrained, with C3+C4 species providing an essential step for C4 evolution.

Respiratory and C4-photosynthetic NAD-malic enzyme coexist in bundle sheath cells mitochondria and evolved via association of differentially adapted subunits

In different lineages of Cleomaceae, NAD-malic enzyme (NAD-ME) was independently co-opted to participate in C4 photosynthesis. In the C4 Cleome species Gynandropsis gynandra and Cleome angustifolia, all NAD-ME genes (NAD-MEα, NAD-MEβ1, and NAD-MEβ2) were affected by C4 evolution and are expressed at higher levels than their orthologs in the C3 Cleome species Tarenaya hassleriana. In the latter C3 species, the NAD-ME housekeeping function is performed by two heteromers, NAD-MEα/β1 and NAD-MEα/β2, with similar biochemical properties. In both C4 species analyzed, this role is restricted the NAD-MEα/β2 heteromer. In the C4 species, NAD-MEα/β1 is exclusively present in the leaves, where it accounts for most of the enzymatic activity. GgNAD-MEα/β1 exhibits high catalytic efficiency and is differentially activated by the C4 intermediate aspartate, confirming its role as the C4-decarboxylase. During C4 evolution, GgNAD-MEβ1and CaNAD-MEβ1 lost their catalytic activity; their contribution to enzymatic activity results from a stabilizing effect on the associated α-subunit. We conclude that in bundle sheath cell mitochondria of C4 Cleome species, the functions of NAD-ME as C4 photosynthetic decarboxylase and as a tricarboxylic acid cycle-associated housekeeping enzyme coexist and are performed by isoforms that combine the same α subunit with differentially adapted β subunits.

STOMAGEN-like Regulates Stomatal Generation Causing Increased Water Use Efficiency during C4 Evolution

Compared with C3 plants, C4 plants maintain lower stomatal conductance (gs), attributed to decreased maximal stomatal conductance (gsmax), without losing photosynthetic CO2 uptake rate. Which stomatal characteristics caused the decreased gsmax and how did the characteristics change along C4 evolution as well as the molecular mechanism underlying this change remains unknown.Stomatal patternings were examined in Flaveria genus, which contains species at different evolutionary stages from C3 to C4 photosynthesis. We further used comparative transcriptomics, transgenic experiments and semi-in-vitro analysis to identify the gene underlying the decreased gsmax in C4 species.Results were as follows: the evolution from C3 to C4 species was accompanied by a stepwise decrease in stomatal density (SD) and dramatic decreased SD occurred between C3-C4 species and C4-like species; FSTOMAGEN gene positively controls SD and its changed expression underlies the decreased SD during C4 evolution; Furthermore, this mechanism is shared between monocotyledons and dicotyledons, indicated by the lower expression of FSTOMAGEN homologs in maize than rice.This study suggests that lower SD is another major feature of C4 evolution, besides C4 enzymes and Kranz anatomy. FSTOMAGEN can be used to engineer lowered SD, a C4 feature required in the current effort of C4 crop engineering.

The legacy of C4 evolution in the hydraulics of C3 and C4 grasses

Most studies concerning the functional implications of C4 photosynthesis have focused on enhanced carbon fixation under high temperature, low atmospheric CO2, and/or water limitation, yet the biochemical and anatomical reorganization required for optimal C4 function should also impact plant hydraulics and water use. C4 grasses have increased bundle-sheath size and vein density, and these are thought to have been anatomical precursors for the evolution of C4 from C3 ancestors. Paradoxically, these traits should also lead to higher leaf capacitance and higher leaf hydraulic conductance, yet C4 photosynthesis lowers water demand and increases plant water use efficiency. Here, we use phylogenetic analyses, physiological measurements and photosynthetic modeling to examine the reorganization of hydraulic traits in C4 grass lineages and in closely-related C3 grasses. Evolutionarily young C4 lineages have higher leaf hydraulic conductance, capacitance, turgor loss point, and lower stomatal conductance than their C3 relatives. In contrast, species from older C4 lineages show decreased leaf hydraulic conductance and capacitance, indicating that over time, C4 plants have optimized hydraulic investments while maintaining their C4 anatomical requirements. The “overplumbing” of young C4 lineages lead to a reduced positive correlation between maximal assimilation rate and leaf hydraulic conductance, decoupling a key relationship between hydraulic traits and photosynthesis generally observed in vascular plants.