Increased α-ketoglutarate links the C3-C4 intermediate state to C4 photosynthesis in the genus Flaveria.

As a complex trait, C4 photosynthesis has multiple independent origins in evolution. Phylogenetic evidence and theoretical analysis suggest that C2 photosynthesis, which is driven by glycine decarboxylation in the bundle sheath cell, may function as a bridge from C3 to C4 photosynthesis. However, the exact molecular mechanism underlying the transition between C2 photosynthesis to C4 photosynthesis remains elusive. Here, we provide evidence suggesting a role of higher α-ketoglutarate (AKG) concentration during this transition. Metabolomic data of 12 Flaveria species, including multiple photosynthetic types, show that AKG concentration initially increased in the C3-C4 intermediate with a further increase in C4 species. Petiole feeding of AKG increases the concentrations of C4-related metabolites in C3-C4 and C4 species but not the activity of C4-related enzymes. Sequence analysis shows that glutamate synthase (Fd-GOGAT), which catalyzes the generation of glutamate using AKG, was under strong positive selection during the evolution of C4 photosynthesis. Simulations with a constraint-based model for C3-C4 intermediate further show that decreasing the activity of Fd-GOGAT facilitated the transition from a C2-dominant to a C4-dominant CO2 concentrating mechanism. All these results provide insight into the mechanistic switch from C3-C4 intermediate to C4 photosynthesis.

Two major metabolic factors for an efficient NADP-malic enzyme type C4 photosynthesis.

Compared to the large number of studies focused on the factors controlling C3 photosynthesis efficiency, there are relatively fewer studies of the factors controlling photosynthetic efficiency in C4 leaves. Here, we used a dynamic systems model of C4 photosynthesis based on maize (Zea mays) to identify features associated with high photosynthetic efficiency in NADP-malic enzyme (NADP-ME) type C4 photosynthesis. We found that two additional factors related to coordination between C4 shuttle metabolism and C3 metabolism are required for efficient C4 photosynthesis: (1) accumulating a high concentration of phosphoenolpyruvate through maintaining a large PGA concentration in the mesophyll cell chloroplast and (2) maintaining a suitable oxidized status in bundle sheath cell chloroplasts. These identified mechanisms are in line with the current cellular location of enzymes/proteins involved in the starch synthesis, the Calvin-Benson cycle and photosystem II of NADP-ME type C4 photosynthesis. These findings suggested potential strategies for improving C4 photosynthesis and engineering C4 rice.

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.

PetM Is Essential for the Stabilization and Function of the Cytochrome b6f Complex in Arabidopsis.

The cytochrome b6f (cyt b6f) acts as a common linker of electron transport between photosystems I and II in oxygenic photosynthesis. PetM, one of eight subunits of the cyt b6f complex, is a small hydrophobic subunit at the outside periphery, the functional mechanism of which remains to be elucidated in higher plants. In this work, we found that unlike the PetM mutant in Synechocystis sp. PCC 6803, the Arabidopsis thaliana PetM mutant showed a bleached phenotype with yellowish leaves, block of photosynthetic electron transport and loss of photo-autotrophy, similar to the Arabidopsis PetC mutant. Although PetM is relatively conserved between higher plants and cyanobacteria, Synechocystis PetM could not rescue the PetM-knockout phenotype in Arabidopsis. We provide evidence that the Synechocystis PetM did not stably bind to the Arabidopsis cyt b6f complex. Based on these results, we suggest that PetM is required by Arabidopsis to maintain the function of the cyt b6f complex, likely through its close link with core subunits to form a tight 'fence' that stabilizes the core of the complex.

Natural variation in the fast phase of chlorophyll a fluorescence induction curve (OJIP) in a global rice minicore panel.

Photosynthesis can be probed through Chlorophyll a fluorescence induction (FI), which provides detailed insight into the electron transfer process in Photosystem II, and beyond. Here, we have systematically studied the natural variation of the fast phase of the FI, i.e. the OJIP phase, in rice. The OJIP phase of the Chl a fluorescence induction curve is referred to as "fast transient" lasting for less than a second; it is obtained after a dark-adapted sample is exposed to saturating light. In the OJIP curve, "O" stands for "origin" (minimal fluorescence), "P" for "peak" (maximum fluorescence), and J and I for inflection points between the O and P levels. Further, Fo is the fluorescence intensity at the "O" level, whereas Fm is the intensity at the P level, and Fv (= Fm - Fo) is the variable fluorescence. We surveyed a set of quantitative parameters derived from the FI curves of 199 rice accessions, grown under both field condition (FC) and growth room condition (GC). Our results show a significant variation between Japonica (JAP) and Indica (IND) subgroups, under both the growth conditions, in almost all the parameters derived from the OJIP curves. The ratio of the variable to the maximum (Fv/Fm) and of the variable to the minimum (Fv/Fo) fluorescence, the performance index (PIabs), as well as the amplitude of the I-P phase (AI-P) show higher values in JAP compared to that in the IND subpopulation. In contrast, the amplitude of the O-J phase (AO-J) and the normalized area above the OJIP curve (Sm) show an opposite trend. The performed genetic analysis shows that plants grown under GC appear much more affected by environmental factors than those grown in the field. We further conducted a genome-wide association study (GWAS) using 11 parameters derived from plants grown in the field. In total, 596 non-unique significant loci based on these parameters were identified by GWAS. Several photosynthesis-related proteins were identified to be associated with different OJIP parameters. We found that traits with high correlation are usually associated with similar genomic regions. Specifically, the thermal phase of FI, which includes the amplitudes of the J-I and I-P subphases (AJ-I and AI-P) of the OJIP curve, is, in turn, associated with certain common genomic regions. Our study is the first one dealing with the natural variations in rice, with the aim to characterize potential candidate genes controlling the magnitude and half-time of each of the phases in the OJIP FI curve.

Knocking out NEGATIVE REGULATOR OF PHOTOSYNTHESIS 1 increases rice leaf photosynthesis and biomass production in the field.

Improving photosynthesis is a major approach to increasing crop yield potential. Here we identify a transcription factor as a negative regulator of photosynthesis, which can be manipulated to increase rice photosynthesis and plant biomass in the field. This transcription factor, named negative regulator of photosynthesis 1 (NRP1; Os07g0471900), was identified through a co-expression analysis using rice leaf RNA sequencing data. NRP1 expression showed significantly negative correlation with the expression of many genes involved in photosynthesis. Knocking out NRP1 led to greater photosynthesis and increased biomass in the field, while overexpression of NRP1 decreased photosynthesis and biomass. Transcriptomic data analysis shows that NRP1 can negatively regulate the expression of photosynthetic genes. Protein transactivation experiments show that NRP1 is a transcription activator, implying that NRP1 may indirectly regulate photosynthetic gene expression through an unknown regulator. This study shows that combination of bioinformatics analysis with transgenic testing can be used to identify new regulators to improve photosynthetic efficiency in crops.

Photosynthetic and transcriptomic responses of two C4 grass species with different NaCl tolerance.

This report reveals the effects of salt on the photosynthetic electron transport and transcriptome of the glycophyte Setaria viridis (S. viridis) and its salt-tolerant close relative halophyte Spartina alterniflora (S. alterniflora). S. viridis was unable to survive exposed to sodium chloride (NaCl) levels higher than 100 mM, in contrast, S. alterniflora could tolerate NaCl up to 550 mM, with negligible effect on gas exchange related parameters and conductance of electrons transport chain (gETC). Under salt, the prompt fluorescence (OJIP-curves) exhibits an increase in the O- and J-steps in S. viridis and much less for S. alterniflora. Flowing NaCl stress, a dramatic decline in the photosystem II (PSII) primary photochemistry was observed for S. viridis, as reflected by the drastic drop in Fv/Fm, Fv/Fo and ΦPSII; however, no substantial change was recorded for these parameters in S. alterniflora. Interestingly, we found an increase in the primary PSII photochemistry (ΦPSII) for S. alterniflora with increasing either NaCl concentration or NaCl treatment duration. The NPQ magnitude was strongly enhanced for S. viridis even at a low NaCl (50 mM); however, it remains unchangeable or slightly increased for S. alterniflora at NaCl levels above 400 mM. After NaCl treatment, we found an increase in both the proportion of oxidized P700 and the amount of active P700 in S. viridis and almost no change for S. alterniflora. Under salt, the net photosynthetic rate (A) and stomatal conductance (gs) measurements demonstrate that A decreases earlier in S. viridis, even after one week exposure to only 50 mM NaCl; in contrast, in S. alterniflora, the effect of NaCl on A and gs was minor even after exposure for two weeks to high NaCl levels. For S. viridis exposed to 50 mM NaCl for 12 d, carbon dioxide (CO2) at a concentration of 2000 µL L-1 could not fully restore A to the control (Ctrl) level. Conversely, in S. alterniflora, high CO2 can fully restore A for all NaCl treatments except at 550 mM. RNA-seq data shows a major impact of NaCl on metabolic pathways in S. viridis and we found a number of transcription factors potentially related to NaCl responses. For S. alterniflora, no major changes in the transcriptomic levels were recorded under NaCl stress. To confirm our data analysis of RNA-seq, we performed quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis for randomly selected four genes for each species (8 genes in total) and we found that our results (up- and/or down-regulated genes) are fully consistent and match well our RNA-seq data. Overall, this study showed drastically different photosynthetic and transcriptomic responses of a salt-tolerant C4 grass species and one salt-sensitive C4 grass species to NaCl stress, which suggests that S. alterniflora could be used as a promising model species to study salt tolerance in C4 or monocot species.

Contrasting Responses of Plastid Terminal Oxidase Activity Under Salt Stress in Two C4 Species With Different Salt Tolerance.

The present study reveals contrasting responses of photosynthesis to salt stress in two C4 species: a glycophyte Setaria viridis (SV) and a halophyte Spartina alterniflora (SA). Specifically, the effect of short-term salt stress treatment on the photosynthetic CO2 uptake and electron transport were investigated in SV and its salt-tolerant close relative SA. In this experiment, at the beginning, plants were grown in soil then were exposed to salt stress under hydroponic conditions for two weeks. SV demonstrated a much higher susceptibility to salt stress than SA; while, SV was incapable to survive subjected to about 100 mM, SA can tolerate salt concentrations up to 550 mM with slight effect on photosynthetic CO2 uptake rates and electrons transport chain conductance (gETC ). Regardless the oxygen concentration used, our results show an enhancement in the P700 oxidation with increasing O2 concentration for SV following NaCl treatment and almost no change for SA. We also observed an activation of the cyclic NDH-dependent pathway in SV by about 2.36 times upon exposure to 50 mM NaCl for 12 days (d); however, its activity in SA drops by about 25% compared to the control without salt treatment. Using PTOX inhibitor (n-PG) and that of the Qo-binding site of Cytb6/f (DBMIB), at two O2 levels (2 and 21%), to restrict electrons flow towards PSI, we successfully revealed the presence of a possible PTOX activity under salt stress for SA but not for SV. However, by q-PCR and western-blot analysis, we showed an increase in PTOX amount by about 3-4 times for SA under salt stress but not or very less for SV. Overall, this study provides strong proof for the existence of PTOX as an alternative electron pathway in C4 species (SA), which might play more than a photoprotective role under salt stress.

What Matters for C4 Transporters: Evolutionary Changes of Phosphoenolpyruvate Transporter for C4 Photosynthesis.

C4 photosynthesis is a complex trait that evolved from its ancestral C3 photosynthesis by recruiting pre-existing genes. These co-opted genes were changed in many aspects compared to their counterparts in C3 species. Most of the evolutionary changes of the C4 shuttle enzymes are well characterized, however, evolutionary changes for the recruited metabolite transporters are less studied. Here we analyzed the evolutionary changes of the shuttle enzyme phosphoenolpyruvate (PEP) transporter (PPT) during its recruitment from C3 to C4 photosynthesis. Our analysis showed that among the two PPT paralogs PPT1 and PPT2, PPT1 was the copy recruited for C4 photosynthesis in multiple C4 lineages. During C4 evolution, PPT1 gained increased transcript abundance, shifted its expression from predominantly in root to in leaf and from bundle sheath cell to mesophyll cell, and gained more rapid and long-lasting responsiveness to light. Modifications occurred in both regulatory and coding regions in C4 PPT1 as compared to C3 PPT1, however, the PEP transporting function of PPT1 remained. We found that PPT1 of a Flaveria C4 species recruited a MEM1 B submodule in the promoter region, which might be related to the increased transcript abundance of PPT1 in C4 mesophyll cells. The case study of PPT further suggested that high transcript abundance in a proper location is of high priority for PPT to support C4 function.

Overexpression of maize transcription factor mEmBP-1 increases photosynthesis, biomass, and yield in rice.

Identifying new options to improve photosynthetic capacity is a major approach to improve crop yield potential. Here we report that overexpression of the gene encoding the transcription factor mEmBP-1 led to simultaneously increased expression of many genes in photosynthesis, including genes encoding Chl a,b-binding proteins (Lhca and Lhcb), PSII (PsbR3 and PsbW) and PSI reaction center subunits (PsaK and PsaN), chloroplast ATP synthase subunit, electron transport reaction components (Fd1 and PC), and also major genes in the Calvin-Benson-Bassham cycle, including those encoding Rubisco, glyceraldehyde phosphate dehydrogenase, fructose bisphosphate aldolase, transketolase, and phosphoribulokinase. These increased expression of photosynthesis genes resulted in increased leaf chlorophyll pigment, photosynthetic rate, biomass growth, and grain yield both in the greenhouse and in the field. Using EMSA experiments, we showed that mEmBP-1a protein can directly bind to the promoter region of photosynthesis genes, suggesting that the direct binding of mEmBP-1a to the G-box domain of photosynthetic genes up-regulates expression of these genes. Altogether, our results show that mEmBP-1a is a major regulator of photosynthesis, which can be used to increase rice photosynthesis and yield in the field.

The transcriptome of NaCl-treated Limonium bicolor leaves reveals the genes controlling salt secretion of salt gland.

Limonium bicolor, a typical recretohalophyte that lives in saline environments, excretes excessive salt to the environment through epidermal salt glands to avoid salt stress. The aim of this study was to screen for L. bicolor genes involved in salt secretion by high-throughput RNA sequencing. We established the experimental procedure of salt secretion using detached mature leaves, in which the optimal salt concentration was determined as 200 mM NaCl. The detached salt secretion system combined with Illumina deep sequencing were applied. In total, 27,311 genes were annotated using an L. bicolor database, and 2040 of these genes were differentially expressed, of which 744 were up-regulated and 1260 were down-regulated with the NaCl versus the control treatment. A gene ontology enrichment analysis indicated that genes related to ion transport, vesicles, reactive oxygen species scavenging, the abscisic acid-dependent signaling pathway and transcription factors were found to be highly expressed under NaCl treatment. We found that 102 of these genes were likely to be involved in salt secretion, which was confirmed using salt-secretion mutants. The present study identifies the candidate genes in the L. bicolor salt gland that are highly associated with salt secretion. In addition, a salt-transporting pathway is presented to explain how Na(+) is excreted by the salt gland in L. bicolor. These findings will shed light on the molecular mechanism of salt secretion from the salt glands of plants.

Transcriptome comparisons shed light on the pre-condition and potential barrier for C4 photosynthesis evolution in eudicots.

C4 photosynthesis evolved independently from C3 photosynthesis in more than 60 lineages. Most of the C4 lineages are clustered together in the order Poales and the order Caryophyllales while many other angiosperm orders do not have C4 species, suggesting the existence of biological pre-conditions in the ancestral C3 species that facilitate the evolution of C4 photosynthesis in these lineages. To explore pre-adaptations for C4 photosynthesis evolution, we classified C4 lineages into the C4-poor and the C4-rich groups based on the percentage of C4 species in different genera and conducted a comprehensive comparison on the transcriptomic changes between the non-C4 species from the C4-poor and the C4-rich groups. Results show that species in the C4-rich group showed higher expression of genes related to oxidoreductase activity, light reaction components, terpene synthesis, secondary cell synthesis, C4 cycle related genes and genes related to nucleotide metabolism and senescence. In contrast, C4-poor group showed up-regulation of a PEP/Pi translocator, genes related to signaling pathway, stress response, defense response and plant hormone metabolism (ethylene and brassinosteroid). The implications of these transcriptomic differences between the C4-rich and C4-poor groups to C4 evolution are discussed.

RNA-Seq based phylogeny recapitulates previous phylogeny of the genus Flaveria (Asteraceae) with some modifications.

BACKGROUND: The genus Flaveria has been extensively used as a model to study the evolution of C4 photosynthesis as it contains C3 and C4 species as well as a number of species that exhibit intermediate types of photosynthesis. The current phylogenetic tree of the genus Flaveria contains 21 of the 23 known Flaveria species and has been previously constructed using a combination of morphological data and three non-coding DNA sequences (nuclear encoded ETS, ITS and chloroplast encoded trnL-F). RESULTS: Here we developed a new strategy to update the phylogenetic tree of 16 Flaveria species based on RNA-Seq data. The updated phylogeny is largely congruent with the previously published tree but with some modifications. We propose that the data collection method provided in this study can be used as a generic method for phylogenetic tree reconstruction if the target species has no genomic information. We also showed that a "F. pringlei" genotype recently used in a number of labs may be a hybrid between F. pringlei (C3) and F. angustifolia (C3-C4). CONCLUSIONS: We propose that the new strategy of obtaining phylogenetic sequences outlined in this study can be used to construct robust trees in a larger number of taxa. The updated Flaveria phylogenetic tree also supports a hypothesis of stepwise and parallel evolution of C4 photosynthesis in the Flavaria clade.

Comparative transcriptome analysis of developmental stages of the Limonium bicolor leaf generates insights into salt gland differentiation.

With the expansion of saline land worldwide, it is essential to establish a model halophyte to study the salt-tolerance mechanism. The salt glands in the epidermis of Limonium bicolor (a recretohalophyte) play a pivotal role in salt tolerance by secreting excess salts from tissues. Despite the importance of salt secretion, nothing is known about the molecular mechanisms of salt gland development. In this study, we applied RNA sequencing to profile early leaf development using five distinct developmental stages, which were quantified by successive collections of the first true leaves of L. bicolor with precise spatial and temporal resolution. Specific gene expression patterns were identified for each developmental stage. In particular, we found that genes controlling salt gland differentiation in L. bicolor may evolve in a trichome formation, which was also confirmed by mutants with increased salt gland densities. Genes involved in the special ultrastructure of salt glands were also elucidated. Twenty-six genes were proposed to participate in salt gland differentiation. Our dataset sheds light on the molecular processes underpinning salt gland development and thus represents a first step towards the bioengineering of active salt-secretion capacity in crops.