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.

The C4 Atriplex halimus vs. the C3 Atriplex hortensis: Similarities and Differences in the Salinity Stress Response

Soil properties and the ability to sustain agricultural production are seriously impaired by salinity. The cultivation of halophytes is seen as a solution to cope with the problem. In this framework, a greenhouse pot experiment was set up to assess salinity response in the perennial C4 species Atriplex halimus, and in the following three cultivars of the annual C3 Atriplex hortensis: green, red, and scarlet. The four genotypes were grown for 35 days with water salinity (WS) ranging from 0 to 360 mM NaCl. Plant height and fresh weight (FW) increased at 360 vs. 0 WS. The stomatal conductance (GS) and transpiration rate (E) were more severely affected by salinity in the C4 A. halimus than in the C3 species A. hortensis. This was reflected in a lower leaf water potential indicating stronger osmotic adjustment, and a higher relative water content associated with more turgid leaves, in A. halimus than A. hortensis. In a PCA including all the studied traits, the GS and E negatively correlated to the FW, which, in turn, positively correlated with Na concentration and intrinsic water use efficiency (iWUE), indicating that reduced gas exchange associated with Na accumulation contributed to sustain iWUE under salinity. Finally, FTIR spectroscopy showed a reduced amount of pectin, lignin, and cellulose under salinity, indicating a weakened cell wall structure. Overall, both species were remarkably adapted to salinity: From an agronomic perspective, the opposite strategies of longer vs. faster soil coverage, involved by the perennial A. halimus vs. the annual A. hortensis cv. scarlet, are viable natural remedies for revegetating marginal saline soils and increasing soil organic carbon.

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.

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.

The Coordination and Jumps along C4 Photosynthesis Evolution in the Genus Flaveria

C4 photosynthesis is a remarkable complex trait, elucidations of the evolutionary trajectory of C4 photosynthesis from its ancestral C3 pathway can help us better understand the generic principles of the evolution of complex trait and guide the engineering of C3 crops for higher yields. Here, we used the genus Flaveria that contains C3, C3-C4, C4-like and C4 species as a system to study the evolution of C4 photosynthesis. We first mapped transcript abundance, protein sequence, and morphological features to the phylogenetic tree of the genus Flaveria, and calculated the evolutionary correlation of different features; we then predicted the relative changes of ancestral nodes of those features to illustrate the key stages during the evolution of C4 photosynthesis. We found that gene expression and protein sequence showed consistent modification pattern along the phylogenetic tree. High correlation coefficients ranging from 0.46 to 0.9 among gene expression, protein sequence and morphology were observed, and the greatest modification of those different features consistently occurred at the transition between C3-C4 species and C4-like species. Our results show highly coordinated changes in gene expression, protein sequence and morphological features, which support an obviously evolutionary jump during the evolution of C4 metabolism.