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.

Challenges and Approaches to Crop Improvement Through C3-to-C4 Engineering

With a rapidly growing world population and dwindling natural resources, we are now facing the enormous challenge of increasing crop yields while simultaneously improving the efficiency of resource utilization. Introduction of C4 photosynthesis into C3 crops is widely accepted as a key strategy to meet this challenge because C4 plants are more efficient than C3 plants in photosynthesis and resource usage, particularly in hot climates, where the potential for productivity is high. Lending support to the feasibility of this C3-to-C4 engineering, evidence indicates that C4 photosynthesis has evolved from C3 photosynthesis in multiple lineages. Nevertheless, C3-to-C4 engineering is not an easy task, as several features essential to C4 photosynthesis must be introduced into C3 plants. One such feature is the spatial separation of the two phases of photosynthesis (CO2 fixation and carbohydrate synthesis) into the mesophyll and bundle sheath cells, respectively. Another feature is the Kranz anatomy, characterized by a close association between the mesophyll and bundle sheath (BS) cells (1:1 ratio). These anatomical features, along with a C4-specific carbon fixation enzyme (PEPC), form a CO2-concentration mechanism that ensures a high photosynthetic efficiency. Much effort has been taken in the past to introduce the C4 mechanism into C3 plants, but none of these attempts has met with success, which is in my opinion due to a lack of system-level understanding and manipulation of the C3 and C4 pathways. As a prerequisite for the C3-to-C4 engineering, I propose that not only the mechanisms that control the Kranz anatomy and cell-type-specific expression in C3 and C4 plants must be elucidated, but also a good understanding of the gene regulatory network underlying C3 and C4 photosynthesis must be achieved. In this review, I first describe the past and current efforts to increase photosynthetic efficiency in C3 plants and their limitations; I then discuss a systems approach to tackling down this challenge, some practical issues, and recent technical innovations that would help us to solve these problems.

The diversification of downy mildew species was not driven by the loss of mycorrhizal associations or the evolution of C4 photosynthesis

There are approximately 700 obligate biotrophic species grouped into 20 genera (Oomycota, Peronosporaceae) that cause downy mildew diseases. Dick hypothesized in 2001 that diversification of downy mildew species was driven, in part, by host plant secondary metabolites. Dick further speculated that this was driven by the transition of host plants away from mycorrhizal associations or the evolution of C4 photosynthesis. Specifically, loss of mycorrhizal associations or the use of C4 photosynthesis would result in more free carbon that the plants could then use to produce more secondary metabolites. If true, then there should be more downy mildew species that infect hosts from plant lineages that lack mycorrhizal associations or use C4 photosynthesis. However, analysis of 677 downy mildew species for host plant mycorrhizal associations and host plant photosynthetic pathway type show that this is not what occurred. Seventy percent of downy mildew species parasitize hosts that form mycorrhizal associations and 94% of downy mildew species parasitize hosts that use C3 photosynthesis. From this, it is concluded that the diversification of downy mildew species was not driven by the loss of mycorrhizal associations or the evolution of C4 photosynthesis. However, 85% of downy mildew species that parasitize Poaceae (grasses) parasitize C4 hosts. Thus, it is possible that C4 photosynthesis plays a role in the diversification of these genera.

The coordination of major events in 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 traits 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 onto 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 major events during the evolution of C4 photosynthesis. We found that gene expression and protein sequence showed consistent modification patterns in the phylogenetic tree. High correlation coefficients ranging from 0.46 to 0.9 among gene expression, protein sequence and morphology were observed. 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 evolutionary major events during the evolution of C4 metabolism.

Developmental Effects on Relative Use of PEPCK and NADP-ME Pathways of C4 Photosynthesis in Maize

C4 photosynthesis is an adaptive photosynthetic pathway which concentrates CO2 around Rubisco in specialized bundle sheath cells to reduce photorespiration. Historically, the pathway has been characterized into three different subtypes based on the decarboxylase involved, although recent work has provided evidence that some plants can use multiple decarboxylases, with maize in particular using both the NADP-malic enzyme (NADP-ME) pathway and phosphoenolpyruvate carboxykinase (PEPCK) pathway. Parallel C4 pathways could be advantageous in balancing energy and reducing equivalents between bundle sheath and mesophyll cells, in decreasing the size of the metabolite gradients between cells and may better accommodate changing environmental conditions or source to sink demands on growth. The enzyme activity of C4 decarboxylases can fluctuate with different stages of leaf development, but it remains unclear if the pathway flexibility is an innate aspect of leaf development or an adaptation to the leaf microenvironment that is regulated by the plant. In this study, variation in the two C4 pathways in maize were characterized at nine plant ages throughout the life cycle. Two positions in the canopy were examined for variation in physiology, gene expression, metabolite concentration, and enzyme activity, with particular interest in asparagine as a potential regulator of C4 decarboxylase activity. Variation in C4 and C3 metabolism was observed for both leaf age and canopy position, reflecting the ability of C4 pathways to adapt to changing microenvironments.

Exploring the Functional Strategies Adopted by Coastal Plants Along an Ecological Gradient Using Morpho-functional Traits

Coastal dunes are characterised by strong interactions between biotic and abiotic factors along a short gradient from the shoreline to the inland region. We carried out an ecological analysis of the vegetation in a protected area of the Italian coast to evaluate the relationships among species abundance, the occurrence of morphoanatomical traits related to leaves, stems, and roots, and soil variables. Three transects were established perpendicular to the shoreline, with 27 plots distributed in the frontal dunes, backdunes, and temporarily wet dune slacks. An analysis based on community-weighted mean values showed that the pioneer communities of the frontal dunes were dominated by ruderals that are well adapted to the harsh ecological conditions of these environments, showing succulent leaves, high limb thickness values, and low values for leaf dry matter content (LDMC). The backdune vegetation was a mosaic of annual herbaceous and perennial shrub communities showing both ruderal and stress-tolerant strategies (clonality, sclerified leaves, high LDMC values, root phenolics) consistent with less extreme ecological conditions. The dune slack areas were dominated by plants showing adaptations to both arid and flooded environments, such as C4 photosynthesis, amphistomatic leaves, and abundant aerenchyma in the roots. The invasive status, C4 photosynthesis, leaf trichomes, and aerenchyma in the roots were significantly correlated with soil humidity, organic matter content, and pH. These results demonstrate the usefulness of anatomical traits (including root system traits) in understanding the functional strategies adopted by plants. Invasive species tended to occupy plots with high levels of soil moisture, suggesting an avoidance strategy for the harsh environmental conditions of coastal sand dunes. Finally, we suggest including information regarding root systems into coastal monitoring programs because they are directly linked to soil parameters useful in coastal dune management and protection.

A synthetic C4 shuttle via the β-hydroxyaspartate cycle in C3 plants

Plants depend on the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) for CO2 fixation. However, especially in C3 plants, photosynthetic yield is reduced by formation of 2-phosphoglycolate, a toxic oxygenation product of Rubisco, which needs to be recycled in a high-flux–demanding metabolic process called photorespiration. Canonical photorespiration dissipates energy and causes carbon and nitrogen losses. Reducing photorespiration through carbon-concentrating mechanisms, such as C4 photosynthesis, or bypassing photorespiration through metabolic engineering is expected to improve plant growth and yield. The β-hydroxyaspartate cycle (BHAC) is a recently described microbial pathway that converts glyoxylate, a metabolite of plant photorespiration, into oxaloacetate in a highly efficient carbon-, nitrogen-, and energy-conserving manner. Here, we engineered a functional BHAC in plant peroxisomes to create a photorespiratory bypass that is independent of 3-phosphoglycerate regeneration or decarboxylation of photorespiratory precursors. While efficient oxaloacetate conversion in Arabidopsis thaliana still masks the full potential of the BHAC, nitrogen conservation and accumulation of signature C4 metabolites demonstrate the proof of principle, opening the door to engineering a photorespiration-dependent synthetic carbon–concentrating mechanism in C3 plants.

Cis-regulatory divergence underpins the evolution of C3-C4 intermediate photosynthesis in Moricandia

Altered transcript abundances and cell specific gene expression patterns that are caused by regulatory divergence play an important role in the evolution of C4 photosynthesis. How these altered gene expression patterns are achieved and whether they are driven by cis- or trans-regulatory changes is mostly unknown. To address this question, we investigated the regulatory divergence between C3 and C3-C4 intermediates, using allele specific gene expression (ASE) analyses of Moricandia arvensis (C3-C4), M. moricandioides (C3) and their interspecific F1 hybrids. ASE analysis on SNP-level showed similar relative proportions of regulatory effects among hybrids: 36% and 6% of SNPs were controlled by cis-only and trans-only changes, respectively. GO terms associated with metabolic processes and the positioning of chloroplast in cells were abundant in transcripts with cis-SNPs shared by all studied hybrids. Transcripts with cis-specificity expressed bias toward the allele from the C3-C4 intermediate genotype. Additionally, ASE evaluated on transcript-level indicated that ~27% of transcripts show signals of ASE in Moricandia hybrids. Promoter-GUS assays on selected genes revealed altered spatial gene expression patterns, which likely result from regulatory divergence in their promoter regions. Assessing ASE in Moricandia interspecific hybrids contributes to the understanding of early evolutionary steps towards C4 photosynthesis and highlights the impact and importance of altered transcriptional regulations in this process.